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
  • PrimePCR™ SYBR® Green Assay: HPRT1, 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 7ml of K562 at 430cells/μl
  • We will follow this protocol for culturing the THP-1 cells.
    • However, keep the THP-1 and K562 cell suspensions separated for this experiment. We want qPCR to only measure the effect on K562 cells.
  • The goal of this experiment is to see if K562 cells can be stimulated by THP-1 Cells through Paracrine signalling.

On Day of Experiment¶

  • Make 6 K562 1.5ml Tubes with DMEM 1%PEN-STREP with 0.5ml of K562 Cells at 430cells/μl in each:
    • We will label these tubes 3hr, 2hr, 1hr, 0hr, No Stimulation and TNFα
    • Keep these tubes with lid open inside an incubator at 37°C and 5%CO2
  • Make enough Lysis reagent for 13 samples and keep on ice.
  • Thaw LPS and TNFα Vials and then keep on ice.
  • Load the ChronoSeq device reservoir 1 with 40ml of THP-1 Cell suspension at 430cells/μl similar to Bulk ChronoSeq however, we will be taking samples manually.

Cell Lysis¶

  • We will follow a protocol similar to SingleShot Cell Lysis Kit but we will not change our media to DPBS.

  • This is what our overall protocol looks like:

    • THP-1 cell suspension stimulated with 1μg/ml LPS.
    • Samples of THP-1 Cells taken at 0hr, 1hr, 2hr and 3hr.
    • Supernatant of THP-1 samples then added to K562 Cell suspension.
    • K562 NFKBIA and ACTB Genes measured at 0hr after adding THP-1 Supernatant, and 1hr after adding THP-1 Supernatant.

  • Prepare 12 PCR Tubes for Cell Lysis as follows and keep these Tubes on ice while your prepare. For each tube:

    • Label the Tubes 3H_0,3H_1,2H_0,2H_1,1H_0,1H_1,0H_0,0H_1,NS_0,NS_1,TNF_0,TNF_1.
    • 48μl Single Shot Cell Lysis Buffer
    • 1μl Proteinase K Solution
    • 1μl DNase Solution

  • For each tube we will:

    • 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 12 samples.

  • We need to take both 0hr and 1hr samples for K562 as well. This will help calculate the impact of LPS stimulation alone as well. We will only take 0.9ml Samples of THP-1 cells to conserve the amount left for our experiment. Always keep the K562 tubes back at 37°C and 5%CO2.

Time THP-1 Sample K562 5μl Sample for qPCR
0hr Add LPS take 0.9 ml sample spin down at 1500g for 1 minute and then add 500μl Supernatant to K562 0hr Epie Immediately also take sample for K562 0H_0
30min Add 0.5ml of DMEM 1%PEN-STREP media to No Stimulation and TNFα Tubes. Immediately add 1μl of TNFα stock to TNFα flask Immediately take 0hr sample for both K562 Tubes NS_0,TNF_0
1hr take 0.9 ml sample spin down at 1500g for 1 minute and then add 500μl Supernatant to K562 1hr flask Immediately take sample from 1hr tube for 1H_0 and then take sample from K562 0hr tube for 0H_1
1hr30mins Do nothing Take samples from No Stimulation NS_1 and TNF&alpha K562 Tube for TNF_1
2hr take 0.9 ml sample spin down at 1500g for 1 minute and then add 500μl Supernatant to K562 2hr flask Immediately take sample from 2hr tube for 2H_0 and then take sample from K562 1hr tube for 1H_1
3hr take 0.9 ml sample spin down at 1500g for 1 minute and then add 500μl Supernatant to K562 3hr flask Immediately take sample from 3hr tube for 3H_0 and then take sample from K562 2hr tube for 2H_1
4hr Do nothing Take sample from K562 3hr tube for 3H_1
  • 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
  • Here is a screenshot of the plate setup.
  • 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.

Additional RT-qPCR for detecting RNAase activity¶

  • To check the effect of RNAase degradation present in THP-1 supernatant we also performed additional qPCR experiments for the low expressed housekeeping gene HPRT1
  • Here is a screenshot of the plate setup.
  • We checked the expression of HPRT1 in K562 cells in four conditions and a blank across two replicates. NS_1,TNF_1,0H_1,2H_1 and Blank
    • With only 4μl of Nuclease Free Water added instead of Cell Lysate for the blank.
  • THP-1 supernatant showed RNAase activity with undetectable HPRT1 compared to TNFα and unstimulated samples.
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