This protocol is deprecated please see the new protocol¶

Protocol for qPCR Checking of K562 Costimulation from THP-1 Cells¶

Reagents to purchase¶

  • SingleShot Cell Lysis Kit, 100 x 50 µl rxns #1725080
  • iTaq™ Universal SYBR® Green One-Step Kit, 100 x 20 µl rxns, 1 ml #1725150
  • PrimePCR™ SYBR® Green Assay: NFKBIA, Human
  • PrimePCR™ SYBR® Green Assay: ACTB, Human

Instruments used:¶

  • Bio-Rad CFX Opus 96

Cell Culture and Cell Preparation¶

  • We will need to culture THP-1 cells and K562 Cells for this experiment.
  • Make sure you have enough K562 Cells on the day of the experiment to make at least 15ml of K562 at 430cells/μl
  • We will follow the Bowdish lab protocol for culturing THP-1 Cells
  • Make complete growth media with 10%FBS, 1%PEN-STREP, and 0.05mM (1X)2-Mercaptoethanol with ATCC Formulation of RPMI 1640.
  • The goal of this experiment is to see if K562 cells can be stimulated by THP-1 Cells through Paracrine signalling.
  • We will use vented Tissue Culture Treated T25 Flasks for this experiment.
  • Four days before the experiment(preferably on a Friday) prepare four T25 flasks with 3ml of THP-1 cells at 430cells/μl.
    • Label these flasks 3hr, 2hr, 1hr and 0hr.
  • Make PMA Stock by adding 1μl of 10mM PMA to 1ml of Culture Media. This will give you a 10μM stock. We need to make the final concentration 100nM for the flasks. So for this purpose we will add 30μl of this stock to each Flask.
  • Now leave the cells to adhere and differenciate and come back on Monday. On Monday we will:
    • Remove the Media and dead cells then wash the cells twice with 3ml of fresh Culture Media.
    • Be careful not to detach and cells. Make sure the final volume is still 3ml.
    • Leave the cells for 24 hours to rest.

On Day of Experiment¶

  • Keep DMEM 1%PEN-STEP in Water bath and Turn UV ON in Cell Culture hood for 15minutes before getting started.
  • Thaw LPS Stock Tube and TNFα Stock Tube and keep on ice.
  • Wash all THP-1 Flasks with Serum Free DMEM 1%PEN-STREP twice.
    • Make sure final volume is still 3ml.
    • Don't detach the cells.
  • We will prepare 6 T25 Flasks of K562 Cells at 430Cells/ul at 2ml each. K562 Cells will be suspended in DMEM 1%PEN-STREP.
    • These will be labeled K562: 3hr, 2hr, 1hr, 0hr, No Stimulation, TNFα
  • Keep all Flasks in 37°C incubator while we wait.
    • Put another flask with 10ml of DMEM 1%PEN-STREP only also in the incubator.
  • Now we will add 3μl of LPS from the stock tube to get 1μg of LPS/ml in the following order:
    • First add to 3hr THP-1.
    • After 1 hour add to 2hr THP-1
    • After another 1 hour add to 1hr THP-1
    • After another 1 hour add to 0hr THP-1
  • We will immediately transfer the following volumes to K562 Flasks:
    • Add 2ml Supernatant only from 3hr THP-1 to K562 3hr
    • Add 2ml Supernatant only from 2hr THP-1 to K562 2hr
    • Add 2ml Supernatant only from 1hr THP-1 to K562 1hr
    • Add 2ml Supernatant only from 0hr THP-1 to K562 0hr
    • Add 2ml DMEM 1%PEN-STREP from Incubator to K562 No Stimulation
    • Add 2ml DMEM 1%PEN-STREP from Incubator to K562 TNFα and then add 4μl of TNFα from the stock to get a final concentration of 10ng/ml.
  • We will now wait for 1 hour before doing the Cell Lysis part for qPCR. This should provide enough time for the K562 cells to respond to the signalling molecules in the Supernatant.
  • You can prepare Cell Lysis Solution stock while you wait. Make enough for 7 Tubes.

Cell Lysis¶

  • We will follow a protocol similar to SingleShot Cell Lysis Kit but we will not change our media to DPBS.
  • Prepare 6 PCR Tubes for Cell Lysis as follows and keep these Tubes on ice while your prepare. For each tube:
    • Label the Tubes 3H,2H,1H,0H,NS,TNF.
    • 48μl Single Shot Cell Lysis Buffer
    • 1μl Proteinase K Solution
    • 1μl DNase Solution
  • For each tube:
    • Add 5μl of Cell Suspension. Mix Well. This is equivalent to 1075 K562 Cells.
    • Keep the tube back on ice till you have collected all 6 samples.
  • Spin down all the Tubes.
    • Using a PCR Machine run the following program:
      • Lid Temperature 105°C
      • Volume 55μl
      • Incubate for 10 min at 21°C
      • Followed by 5 min at 37°C,
      • 5 min at 75°C.
      • 4°C for ∞
    • The cell lysate can be stored on ice for up to 4 hr, at -20°C for up to 2 months, or at -80°C for up to 12 months.
    • After PCR Immediately store the Tubes at -80°C

Single Step RT-qPCR¶

  • Adapted from existing protocol here.
  • The iTaq kit comes with ROX as a background reference dye so remember to set that in your qPCR machine. ROX is not necessary for Bio-Rad CFX Opus 96.
  • For each conditon you will have to setup 4 qPCR reactions. Two replicates for each gene NFKBIA and ACTB. For each 20μl reaction PCR Tube:
    • iTaq™ Universal SYBR® Green reaction mix 2X: 10μl
    • 1μl PrimePCR Assay primers (20X).
    • iScript reverse transcriptase 0.25μl
    • 4μl Cell Lysate.
    • 4.75μl Nuclease Free Water
  • Setup the qPCR reaction as follows:
    • 10min at 50°C for Reverse Transcription.
    • 1min at 95°C for DNA denaturation and Polymerse activation.
    • 40 Cycles of:
      • 95°C 10seconds Denaturation
      • 60°C 30seconds Annealing and Plate Read
  • After the plate setup. The plate was sealed with a Optically clear adhesive film.
    • Make sure the lips are completely sealed to avoid leaks. Then vortex the plate.
  • Plate was then spun down and loaded into the qPCR machine.
  • Run the Program.