Protocols for Preparing Cells and associated reagents

Equipment Used:

• Our lab uses the refrigerated Sorval ST8R Centrifuge. You can find CAD files to print some of the parts here.
  • We bought the 50ml Inserts and Printed the Rest.
  • You can modify the CAD files to print the 50ml Inserts as well.
  • Use PLA or TPU for the prints.
• CELLTREAT Pipette Controller
  • Replacement Filters
• Eppendorf Pipettes 6-Pack
• Pipette Tips:
  • Genesee Scientific 200/20μl Tips
  • Rainin 1000μl Tips
  • Genesee Scientific 10μl Tips
• Larger Volume Pipettes:
  • 5ml Pipette
  • 10ml Pipette
  • 25ml Pipette
  • 50ml Pipette
• Disposable Sterile Falcon Tubes:
  • 50ml Tubes
  • 15ml Tubes
• Disposable Sterile 250ml GL45 Bottles, Individually Wrapped

Preparation for Starting The Cell Culture

• I found this Playlist very helpful. I recommend watching all these videos before you start.
• At least 7 days before the experiment start your cell culture with U937,EL4, THP-1 or K562 cells.
• Its good to have a large number of preserved stocks for Future Experiments.
• We started the culture from premade Cell stocks preserved in Complete Growth Media and 7% DMSO.
• The Complete Growth Media used for all Cells lines was DMEM with 10% Fetal Bovine Serμm (FBS) and 1% Pen-Strep.
• For the main experiments we also prepared DMEM with 1% Pen-Strep Only.
• All the Media was made using Aseptic Technique inside a Cell Culture Hood.
• Aseptically also make 50μl Aliquots of 10 mg/ml Puromycin Solution for later use.

Protocol for Starting Cell Culture:

• Disinfect the Cell Culture Hood with 70% Ethanol and Turn on the UV.
• Preheat your Complete Growth media(Eg. DMEM with 10%FBS and 1%PEN-STREP) in a water bath set to 37°C. Wait for 15 minutes.
• Aseptically add 9 ml of warm media to 15ml Tubes labeled with the HEK293 and 3T3 or K562.
• Now quickly thaw your preserved Vials of U937,EL4 or K562 in the water bath.
  • Shake the Cells in the Water Bath till all the Ice has disappeared.
• Spray down the Vials throughly with 70% Ethanol.
• Aseptically, transfer the Liquid in the Vials to their labeled 15ml Tube.
• Spin down the Cells at 500xg for Five Minutes. Spray with 70% Ethanol.
• Carefully remove the supernatant without disturbing the Cell Pellets.
• Spray your hands with Ethanol between handling each Cell line.
• Resuspend the Cells in 5ml of Complete Growth Media.
• Now Transfer the Cells to labelled Cell Culture Flasks. Label the Flasks with your Name, the Date, the Cell line name and the Media used to Grow the Cells.
• Add additional 30ml of Complete Growth Media to each flask.
• Put the Flasks in the incubator for four days and keep checking the cells before you start the experiments.

Protocol for Preserving Adherent Cells such as 3T3 and HEK293 in DMSO:

• Put the Complete Growth Media (DMEM with 10%FBS and 1%PEN-STREP), DPBS, and TrypLE express bottles in the 37°C water bath in the Cell culture room.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• Examine your cells under the microscope for Health and Contamination.
• Aseptically remove the Media in the Cell Culture Vessels for 3T3 and HEK293.
• Wash the Cells gently with 10ml of DPBS twice.
• Add 5 ml of TrypLE express to both culture vessels. Make sure there is an even coat on the Cells.
• Place in the incubator for 5 minutes at 37°C.
• Remove the cells and check below the Culture Vessel to see if the Cells are detached completely.
• Swirl the Culture Vessels in Circles to detach all the Cells.
• If the Cell density is very high it might be difficult to break the cells into a single-cell suspension. Put the Cells back into the incubator for an additional 3 minutes of digestion in this case.
• Pipette the Cells up and down using a 5ml pipette to break any remaining chunks.
• Aseptically Transfer the Cells to two 15ml Tubes labeled with the name of the Cell Line on each tube.
• Spin down at 500g for 5 minutues.
• Remove the TrypLE express without disturbing the Cell pellets.
• Aseptically Add 10 ml of Complete Growth Media and resuspend the Cells by pipetting up and down.
• Using Sterile Tips Aseptically Add 700μl of DMSO (472301-100ML) to the Cell Suspension and pipette up and down to mix well.
• Using Sterile Tips Transfer 1ml of this mixture to 10 Sterile Cell preservation Cryo Vials.
  • Label each Vial with your name, the Date and the name of the Cell Line.
• Add these Vials to a Cryopreservation Container.
  • Make sure the Isopropanol(IPA) in the container is fresh. IPA should be replaced after 5 uses.
• Leave the Lid ofthe Cryoperservation Container slightly loose.
• Put this container inside the -80°C freezer.
• Wait 24 hours and then quickly transfer these Cells to your Cell Storage Box in -80°C and/or Liquid Nitrogen Vapor Phase.

Protocol for Preserving Suspension Cells such as K562 in DMSO:

• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• Examine your cells under the microscope for Health and Contamination.
• Aseptially Spin down all your Cells in a Sterile 50ml Tube.
• Remove all the media without disturbing the Cell Pellet.
• Evenly Resuspend the Cells in 10ml of DMEM with 1% Pen-Strep and 10% Fetal Bovine Serum (FBS).
• Aseptically Transfer 10ml Cell Suspension to a 15ml Falcon Tube labeled with the name of the Cell Line.
• Using Sterile Tips Aseptically Add 700μl of DMSO (472301-100ML) to the Cell Suspension and pipette up and down to mix well.
• Using Sterile Tips Transfer 1ml of this mixture to 10 Sterile Cell preservation Cryo Vials.
  • Label each Vial with your name, the Date and the name of the Cell Line.
• Add these Vials to a Cryopreservation Container.
  • Make sure the Isopropanol(IPA) in the container is fresh. IPA should be replaced after 5 uses.
• Leave the Lid ofthe Cryoperservation Container slightly loose.
• Put this container inside the -80°C freezer.
• Wait 24 hours and then quickly transfer these Cells to your Cell Storage Box in -80°C and/or Liquid Nitrogen Vapor Phase.

Protocol for Preparing LPS Solution

• Work aseptically inside the Cell Culture Hood.
• To a Syringe add a 23g Needle.
  • Suck up 1ml of Air into the Syringe
• Prepare a Sterile Eppendorf tube with 1ml of Sterile DPBS.
• Suck up 1ml of DPBS from the Eppendorf tube into the Syringe after this.
• Now insert the Syringe Needle into the LPS Vial and inject all the Liquid into the Vial.
• Raise the Needle so it is not dipped into the Liquid and allow it to suck up some of the Air inside the Vial.
  • The additional Air pressure inside the Vial will force the air into the Syringe.
• Once the pressure has equalized, remove the Syringe Needle from the Vial.
• Shake the Vial to mix the DPBS and LPS evenly.
  • You should now have 1mg/ml of LPS dissolved in the DPBS.
• Vortex the Vial at full speed to evenly mix the LPS.
• Prepare an eppendorf tube for depositing the 1mg/ml LPS Solution.
• Using a new Syringe and Needle suck up 2ml of Air inside the Cell Culture hood.
• Inject the air inside the LPS Vial while keeping the Syringe needle immersed in the liquid.
  • The additional air pressure inside the Vial will push the Liquid inside the Vial into the Syringe.
• Remove the 23g Needle from the Syringe without spilling any liquid. Keep the syringe opening facing up when you do this.
• Add a 5μm Syringe Filter to the Syringe.
• Push the LPS Solution through the Syringe Filter into the Eppendorf tube you prepared earlier.
• Make 50μl Aliquots and store at -20°C.

Protocol for Preparing TNF-α Solution:

• Work aseptically inside the Cell Culture Hood.
• Add 1ml of Sterile DPBS to the TNF-α Vial.
• Vortex at full speed to mix well.
• Spin down the Tube.
• Inside the Cell Culture hood: To a Syringe add a 23g Needle.
• Prepare a second Sterile Eppendorf tube.
• Suck up the 1ml of TNF-α solution from the Eppendorf tube into the Syringe after this.
• Remove the 23g Needle from the Syringe without spilling any liquid. Keep the syringe opening facing up when you do this.
• Add a 5μm Syringe Filter to the Syringe.
• Push the TNF-α Solution through the Syringe Filter into the Eppendorf tube you prepared earlier.
• Make 50μl Aliquots and store at -20°C.

Protocol for Preparing Adherent Cells such as 3T3 and HEK for Production Experiments:

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only, DMEM with 1% PEN-STEP and 10%Fetal Bovine Serum(FBS) DPBS, and TrypLE express bottles in the 37°C water bath in the Cell culture room.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM with 1%PEN-STEP Only depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1%PEN-STREP with a 40μm Cell Strainer
• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

Cell Suspension Preparation and Regrowing Cell Surface Receptors

• Examine your cells under the microscope for Health and Contamination.
• Aim for 80% Confluence of the Cells by the Time you Harvest them.
  • This reduces the chances of larger clumps forming.
• Remove the Media in the Cell Culture Vessels for 3T3 or HEK293.
• Wash the Cells gently with 10ml of DPBS two times.
  • This step is to get rid of all the FBS(Fetal Bovine Serum).
  • When washing make sure you don't detach the cells. Avoid detachment by not directly dispensing liquid over the cells.
• Add 5 ml of TrypLE express to both culture vessels. Make sure there is an even coat on the Cells.
• Place in the incubator for 5 minutes at 37°C.
• Remove the cells and check below the Culture Vessel to see if the Cells are detached completely.
• Swirl the Culture Vessels in Circles to detach all the Cells.
• If the Cell density is very high it might be difficult to break the cells into a single-cell suspension. Put the Cells back into the incubator for an additional 3 minutes of digestion in this case.
• Pipette the Cells up and down using a 5ml pipette to break any remaining chunks.
• Transfer the Cells to two 15ml Tubes labeled with the name of the Cell Line on each tube.
• Spin down at 500g for 5 minutues.
• Remove the TrypLE express without disturbing the Cell pellets.
• Using a 5ml Pipette, resuspend in 5ml of DMEM with 10%FBS and 1% PEN-STEP.
• Now add another 5ml each to the 15ml tubes.
• Label one 50ml Tube with the name of the Cell line.
• Filter the cells using a 40μm Cell strainer

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• Spray this Magnetic Stirring Disk with 70% Ethanol.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean Magnetic Stirring Disk to another 50ml Falcon Tube and add 30ml of DMEM with 10%FBS and 1% PEN-STEP to it. Label this Tube as : HEK293 Suspension for Regrowth of Cell Surface Proteins
• Add the Filtered HEK293 Cell Suspension to this new tube and mix well with the pipette.
• Load this suspension into Cell Reservoir 1 and execute keep_suspension() schedule for 5 hours here).

Make a FBS-Free Cell Suspension after 5 hours

• Freshly Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Get the Cell Suspension from the Cell Reservoir 1. Replace the Tube with the DMEM-1% PEN-STREP that was loaded earlier.
• Inside the Cell Culture hood use a Magnet to Remove the magnetic stirring disk from the Cell Suspension.
• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel. + Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Spin down at 500g for 5 minutues.
• Wash the Cells Thrice using DMEM-1% PEN-STREP:
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 10 ml of DMEM-1% PEN-STREP.
• Filter the cells using a 40μm Cell strainer.
• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 600 cells/μl for Bulk or 215 cells/μl for Single-Cell. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 40ml of HEK293 at 600cells/μl for Bulk Time-Series or 215 cells/μl for Single-Cell Experiments in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tube with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP, Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

• Add the Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 40ml of HEK293 suspension to it. Label this Tube as : HEK293 Suspension, Cell Reservoir 1, 40ml Total Volume

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing Adherent Cells such as 3T3 and HEK for QC Experiments:

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only, DPBS, and TrypLE express bottles in the 37°C water bath in the Cell culture room. Remember we need NO Fetal Bovine Serum (FBS) in our media for these experiments.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM-1% PEN-STREP depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

✶ Note: You don't need to prepare this media if you won't be using the ChronoSeq Device for your experiment.

Cell Suspension Preparation

• Freshly Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Examine your cells under the microscope for Health and Contamination.
• Aim for 80% Confluence of the Cells by the Time you Harvest them.
  • This reduces the chances of larger clumps forming.
• Remove the Media in the Cell Culture Vessels for 3T3 and HEK
• Wash the Cells gently with 10ml of DPBS two times.
  • This step is to get rid of all the FBS(Fetal Bovine Serum).
  • When washing make sure you don't detach the cells. Avoid detachment by not directly dispensing liquid over the cells.
• Add 5 ml of TrypLE express to both culture vessels. Make sure there is an even coat on the Cells.
• Place in the incubator for 5 minutes at 37°C.
• Remove the cells and check below the Culture Vessel to see if the Cells are detached completely.
• Swirl the Culture Vessels in Circles to detach all the Cells.
• If the Cell density is very high it might be difficult to break the cells into a single-cell suspension. Put the Cells back into the incubator for an additional 3 minutes of digestion in this case.
• Pipette the Cells up and down using a 5ml pipette to break any remaining chunks.
• Transfer the Cells to two 15ml Tubes labeled with the name of the Cell Line on each tube.
• Spin down at 500g for 5 minutues.
• Remove the TrypLE express without disturbing the Cell pellets.
• Using a 5ml Pipette, resuspend in 5ml of DMEM-1% PEN-STREP. Use DMEM-1% PEN-STREP for the remaining steps as well from now onwards.
• Now add another 5ml each to the 15ml tubes.
• Label two 50ml Tubes with the name of the Cell lines.
• Filter the cells using a 40μm Cell strainer.

Optional. If your cells are still forming clumps you can do an additional Filtration using a 15&um Pluriselect Filter, Connector Ring and a 50ml Syringe to pull the Cells through using Vacuum from the Syringe. Please see this video from Pluriselect on how to do this.

• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 215 cells/μl for both 3T3 and HEK. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 50ml of 3T3 and 50ml of HEK @ 215 cells/μl in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tubes with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP , Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

For Species Mixing Experiment Using ChronoSeq Device:

• Make 50ml of a 50:50 Mix of 3T3 and HEK293 Cells by mixing 25ml of 3T3 and 25ml of HEK293 made in the previous step.
• If you are running three timepoints for QC you need another two separate tubes of 25ml HEK293 only, and 25ml 3T3 only.
  • Label the first tube as: HEK293 Only Suspension, Cell Reservoir 2, 25ml Total Volume.
  • Label the second tube as: 3T3 Only Suspension, Cell Reservoir 3, 25ml Total Volume.
• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 35ml of 50:50 Mix to it. Label this Tube as : 3T3:HEK 50:50 Mix, Cell Reservoir 1, 35ml Total Volume

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing Suspension Cells such as K562

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only bottle in the 37°C water bath in the Cell culture room. Remember we need NO Fetal Bovine Serum (FBS) in our media for these experiments.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM-1% PEN-STREP depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer

Always make DMEM-1% PEN-STREP Fresh before the Experiment. Do not make Stocks in Advance!

• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

✶ Note: You don't need to prepare this media if you won't be using the ChronoSeq Device for your experiment.

Cell Suspension Preparation

• Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Examine your cells under the microscope for Health and Contamination.
• Aseptically transfer your cells to a Sterile 50ml Falcon Tube.
• Spin down at 500g for 5 minutues.
• Wash the Cells Thrice using DMEM-1% PEN-STREP:
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 10 ml of DMEM-1% PEN-STREP.
• Filter the cells using a 40μm Cell strainer.
• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 215 cells/μl for K562. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 50ml of K562 at 215 cells/μl in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tube with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP, Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

For Experiment Using ChronoSeq Device:

• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 40ml of K562 suspension to it. Label this Tube as : K562 Suspension, Cell Reservoir 1, 40ml Total Volume

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing Peripheral Blood Mononuclear Cells (PBMCs) for Single Cell Experiment

We used this publication as a guide for developing this protocol.

Storage

• Store the PBMCs in Liquid Nitrogen as soon as they arrive. Store at the Bottom of the Dewar.

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only and the DMEM with 1% PEN-STREP 10% FBS (Complete Growth Media) bottles in the 37°C water bath in the Cell culture room. Remember we need NO Fetal Bovine Serum (FBS) in our media for these experiments. However, we need the Complete Growth Media when reviving the Cryopreserved Cells.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM-1% PEN-STREP depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer

Always make DMEM-1% PEN-STREP Fresh before the Experiment. Do not make Stocks in Advance!

• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

✶ Note: You don't need to prepare this media if you won't be using the ChronoSeq Device for your experiment.

Cell Suspension Preparation

• PBMCs are Human Blood products. Please follow the safety protocols for Handling Human Blood and Fluids.
  • For UC San Diego make sure you complete the Blood Borne Pathogen Training.
  • Use a Face Sheild if necessary.
• Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Aseptically Filter 45ml of Complete Growth Media with a 40μm Cell Strainer.
• Transfer the Filtered Media to a Cell Culture Flask.
• Transfer the Flask to an 5% CO₂ incubator set to 37°C.
  • Leave inside the incubator for at least 5 minutes before going to the next step.
• Remove your PBMCs from the Liquid Nitrogen Vapor Phase.
• Quickly thaw inside the 37°C Water bath
  • Avoid immersing the Cap of the Vial below water level to avoid contamination.
  • Gently Shake the Vial to melt the ice.
  • Should be thawed in a few minutes.
• Spray down the Vial with 70% Ethanol and Transfer to the Cell Culture Hood.
• Remove the Cell Culture Flask from the Incubator.
  • Spray it down with 70% Ethanol.
  • Put it inside the Cell Culture Hood.
• Transfer the Thawed Cells to the Cell Culture Flask.
• Wash the PBMC Vial with Warm Complete Growth Media to get any remaining cells out.
  • Transfer this Washing Complete Growth Media to the Cell Culture Flask.
• Transfer the Flask to an 5% CO₂ incubator set to 37°C.
  • Leave inside the incubator for at least 5 minutes before going to the next step.
• Aseptically transfer your cells to a Sterile 50ml Falcon Tube.
• Spin down at 500g for 5 minutues.
• Wash the Cells Thrice using Warm DMEM-1% PEN-STREP:
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 10 ml of DMEM-1% PEN-STREP.
• Filter the cells using a 40μm Cell strainer.
• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 215 cells/μl for the PBMCs. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 50ml of PBMCs at 215 cells/μl in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tube with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP, Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

For Experiment Using ChronoSeq Device:

• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 40ml of PBMC suspension to it. Label this Tube as : PBMC Suspension, Cell Reservoir 1, 40ml Total Volume

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing Suspension Cells such as K562 and EL4 for QC and 3 Timepoint experiment with TNFα Stimulation

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only bottle in the 37°C water bath in the Cell culture room. Remember we need NO Fetal Bovine Serum (FBS) in our media for these experiments.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM-1% PEN-STREP depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer

Always make DMEM-1% PEN-STREP Fresh before the Experiment. Do not make Stocks in Advance!

• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

✶ Note: You don't need to prepare this media if you won't be using the ChronoSeq Device for your experiment.

Cell Suspension Preparation

• Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Examine your cells under the microscope for Health and Contamination.
• Aseptically transfer your cells to a Sterile 50ml Falcon Tube.
• Spin down at 500g for 5 minutues.
• Wash the Cells Thrice using DMEM-1% PEN-STREP:
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 10 ml of DMEM-1% PEN-STREP.
• Filter the cells using a 40μm Cell strainer.
• Additional Filtration. Some EL4 Cells can form clumps and K562 can also be much larger in size. To make the size of the cells similar and to reduce clumps:
  • You can do an additional Filtration using a 15μm Pluriselect Filter, Connector Ring and a 50ml Syringe to pull the Cells through using Vacuum from the Syringe. Please see this video from Pluriselect on how to do this.
  • Filter K562 twice with the 15μm Pluriselect Filter. EL4 Once.
• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 215 cells/μl for K562 and EL4. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 50ml of K562 and EL4 at 215 cells/μl in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tube with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP, Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

• Make 50ml of a 50:50 Mix of K562 and EL4 Cells by mixing 25ml of K562 and 25ml of EL4 made in the previous step.
• If you are running three timepoints for QC you need another two separate tubes of 25ml K562 only, and 25ml EL4 only.
  • Label the first tube as: K562 Only Suspension, Cell Reservoir 2, 25ml Total Volume.
  • Label the second tube as: EL4 Only Suspension, Cell Reservoir 3, 25ml Total Volume.
• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 35ml of K562:EL4 50:50 Mix suspension to it.
  • Label this Tube as : K562:EL4 50:50 Suspension, Cell Reservoir 1, 35ml Total Volume.

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing Suspension Cells such as K562 and U397 for Single Cell Cell Line mixing experiment with LPS Stimulation

Prepare Media For Priming and Flushing Cell Channel of Chrono-Seq Device

• Put the DMEM with 1% PEN-STEP Only bottle in the 37°C water bath in the Cell culture room. Remember we need NO Fetal Bovine Serum (FBS) in our media for these experiments.
• Turn on the UV for the Cell cutlure hood and wait 15 minutes before you start.
• You need about 5ml of media for each timepoint or injection. So for 16 Timepoints you will need about 80ml combined for the Flush containers. Increase this volume for experiments with more timepoints/injections.

• Inside the Cell Culture Hood. Prepare between 100-150ml of DMEM-1% PEN-STREP depending on the number of timepoints/injections for your experiment.
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer

Always make DMEM-1% PEN-STREP Fresh before the Experiment. Do not make Stocks in Advance!

• Label and Transfer Filtered Liquid to 50ml Tubes for Chrono-Seq Device:
  • Label two Sterile/Particle-Free 50ml Falcon Tubes: Cell Media Flush Reservoir: DMEM-1% PEN-STREP
  • Transfer between 25ml-50ml each to the two 50ml Falcon Tubes depending on number of timepoints/injections for the Cell Media Flush Reservoirs.
  • Label three separate Sterile/Particle-Free 50ml Falcon Tubes Cell Sample Reservoir 1: DMEM-1% PEN-STREP, Cell Sample Reservoir 2: DMEM-1% PEN-STREP, and Cell Sample Reservoir 3.: DMEM-1% PEN-STREP
  • Transfer 15ml each to these three 50ml Falcon Tubes for the Cell Sample Reservoirs.
• Store these 5 Falcon Tubes in Tube racks on your Bench for loading into the Chrono-Seq Device.

✶ Note: You don't need to prepare this media if you won't be using the ChronoSeq Device for your experiment.

Cell Suspension Preparation

• Prepare enough Volume of DMEM-1% PEN-STREP for the remaining steps below. To prepare DMEM-1% PEN-STREP:
  • Filter this DMEM-1% PEN-STREP with a 40μm Cell Strainer
• Examine your cells under the microscope for Health and Contamination.
• Aseptically transfer your cells to a Sterile 50ml Falcon Tube.
• Spin down at 500g for 5 minutues.
• Wash the Cells Thrice using DMEM-1% PEN-STREP:
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 25 ml of DMEM-1% PEN-STREP.
  • Spin down at 500g for 5 minutes.
  • Remove most of the media in the Falcon Tube without Disturbing the Cell Pellet.
  • Resuspend the Cells in 10 ml of DMEM-1% PEN-STREP.
• Filter the cells using a 40μm Cell strainer.
• Pipette up and down to make sure the cells have a uniform concentration. Using a P1000, make a 1/20 Dilution using Sterile Tips in a 1.5ml Eppendorf Tube. Do this by adding 50μl of Cell Suspension to 950μl of DMEM-1% PEN-STREP. Make a 1/3 or 1/10 Dilution if your Cell Concentration is not high enough. As a rule of thumb counting less than 100 cells gives a poor estimate of the Initial Undiluted Cell Concentration.
• Mix the 1/20 Dilution throughly using the P1000 by pipetting up and down at least 5 times. This is to make sure the Cells have a uniform concentration. Now you can count the cells.
• Add 20μl of the 1/20 Dilution using a P20 to a Fuchs-Rosenthal Hemocytometer. Count at least 5 squares of the Fuchs-Rosenthal Hemocytometer.
• We need a final concentration of 215 cells/μl for K562. Make the Calculation for getting this final concentration.
• Since each Square of the Hemocytometer is 0.2μl. Five squares should give you the Concentration in the 1/20 Dilution per μl. Multiply this number by 20 to get the Approximate concentration in your Cell Stock in cells/μl.
• Make 50ml of K562 and U937 at 430 cells/μl in DMEM-1% PEN-STREP. Calculate how much volume of cells is needed to make the this final dilution. Make sure the cells have a uniform concentration before making this final dilution.
• Remember to Label the Tube with the Name of the Cell Line, Date, Media:DMEM-1% PEN-STREP, Concentration of Cells, and Volume.

✶ OPTIONAL NOTE: You can make the Same Calculation for a Different Cell Concentration and Volumes if you wish.

• Make 50ml of a 50:50 Mix of K562 and U937 Cells by mixing 25ml of K562 and 25ml of U937 made in the previous step.
• Throughly Clean the Magnetic Stirring Disk with 70% Ethanol.
• Dry this Magnetic Stirring Disk using Particle Free Cleanroom Wipes or Paper Towels.
• Keep this Magnetic Stirring Disk inside the Cell Culture Hood on a Cleanroom wipe or Paper Towel.
• Use the Compressed Air Gun to Blow any Particles off this Magnetic Stirring Disk.
• Add a Clean/Particle Free Magnetic Stirring Disk to another 50ml Falcon Tube and add 40ml of K562:U937 50:50 Mix suspension to it.
  • Label this Tube as : K562:U937 50:50 Suspension, Cell Reservoir 1, 40ml Total Volume.

Location of Magnetic Stirrers	Pouch with Magnetic Stirrers

• You are now ready to run the Machine.

Protocol for Preparing THP-1 cells after PMA differenciation with LPS Stimulation

• We will need to culture THP1 cells and K562 Cells for this experiment. K562 will be prepared similar to previous protocols mentioned above.
• We need to differenciate THP1 cells before we can do a costimulation experiment with K562 and THP1.
• Follow aseptic technique and work inside the cell culture hood. Use sterile tips and pipettes.
• We will need PMA to get started. Abcam provides us with a 10mM solution in DMSO.
  • Make 5μl Aliquots and store at -20°C
• THP1 cells should be adapted to and cultured in Suspension in DMEM with 10% FBS and 1%PEN-STREP (Complete Growth Media).
• Culture THP1 cells in T225 Tissue Culture Treated Vented Flasks. They will provide enough suface area for our THP1 cells to adhere to.
• Have at least 200ml of THP1 cells at a density of 1000-1500 cells per μl.
• We need four T225 Flasks with THP1 cells at a density of 500cells/μl and a total volume of 100ml.
• 40μm Filter cells and transfer into four Sterile 50ml Falcon Tubes.
• Spin down the cells at 500xg for 5 minutes.
• Remove the supernatant and resuspend in fresh complete growth media.
• Add the cells to four T225 flasks and make the final volume 100ml at 500cells/μl
• Next add 1μl of 10mM PMA to each T225 Flask using a P2.5 Pipette. This gives us a final concentration of 100nM PMA.
  • Rock the flasks in a nutating pattern to mix well.
• We will now leave the THP1 cells to differenciate for 48hours. Don't disturb the cells during this period. Keep them at the back of the incubator at 37°C and 5%CO₂ so they are not accidently distubed. During these 48hours our cells will become adherent.
• After 48hours the cells should have a nice monolayer at the bottom of the flask and the supernatant should appeach mostly clear. If you see clumping or colonies of THP1 cells then you might need to reduce the cell concentration and try again.
• Now wash all the walls of the flasks and cells twice with 25ml DPBS with no calcium and magnesium to remove all the PMA.
• Next add 25ml of Complete Growth Media to each flask and let the cells rest without being disturbed for 72hours.

THP-1 Cell Suspension before differenciation	THP-1 Adherent Cells after 48hours of differenciation and 24 hours of rest

• After 72hours the supernatant should look slightly cloudy from some of the cells detaching. Morover, the cells at the bottom of the flasks should appear rounder and less flat.
• We now need to create a Single-Cell Suspension of THP1 cells for our experiment. After which we will do a 50:50 K562:THP1 mix which we will stimulate with 1μg/ml of LPS.
• We will need to make 10mM EDTA stock in DPBS with no calcium and magnesium.
• Put the 10mM EDTA-DPBS stock, Complete Growth Media, and DMEM with 1%PEN-STREP only in the water bath at 37°C to preheat.
• Wash the cells twice with DPBS with no calcium and magnesium.
• Now add 10ml of preheated 10mM EDTA in the DPBS stock to each flask.
  • Make sure there is an even coating on the cells.
• Put the cells in the incubator. Set a timer for 5 minutes.
• Shake the flasks many times to help dislodge the cells. Now put the cells back in the incubator and set another timer for 5 minutes.
• Shake the Flasks again. Now inside the cell culture hood, add 40ml of Complete Growth Media to Neutralize the EDTA.
• At the same time start preparing your K562 Cell Suspension according to the protocol above.
• Spin down the cells at 500xg for 5 minutes at room temperature.
• Remove the supernatant and then resuspend the cell pellet in 10ml of DMEM with 1%PEN-STREP only.
• Combine the cells into a single 50ml Falcon Tube.
• Spin down the cells again.
• Remove the supernatant and resuspend the cell pellet in 25ml of DMEM with 1%PEN-STREP only. You will need to pipette up and down several times to break apart the cell pellet.
• Spin down the cells again.
• Remove the supernatant and resuspend the cells pellet in 10ml of DMEM with 1%PEN-STREP only. You will need to pipette up and down several times to break apart the cell pellet.
• Filter the cells twice with a 40μm Falcon Cell Strainer.
• Make a 1ml 1/10 Dilution and count the cells.
• Make a final stock of 430cells/μl in 40ml.
• Now add a Magnetic Stirring disk to a 50ml Tube and mix 20ml each of K562 and THP1 single cell suspensions at 430cells/μl
• Load the cells into the ChronoSeq device Reservoir 1 as explained previously.