Pluripotency Marker Staining for Flow Cytometry
ReagentVendorCatalog Number
Falcon™ Polystyrene Microplates Fisher Scientific Various
Human Pluripotent Stem Cell Transcription Factor Analysis Kit BD Biosciences 560589
Corning™ Cell Culture PBS (1X) Thermo Fisher / Life Technologies MT21040CV
Accutase Innovative Cell Technologies AT-104
Fisherbrand™ Premium Microcentrifuge Tubes; 1.5 mL Fisher Scientific 05-408-129
Thermo Scientific Thermo Fisher / Life Technologies Various
Corning™ Falcon™ Test Tube with Cell Strainer Snap Cap Fisher Scientific 08-771-23
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Microcentrifuge
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Prepare:
    DMEM/10% FBS (500 mL)
    • DMEM - 450 mL
    • FBS - 50 mL
    hESC (1000 mL)
    • DMEM/F12 – Fill up to 1000 mL
    • KoSR – 200 mL
    • MEM-NEAA – 10 mL
    • L-glutamine – 5mL
    • 2-mercaptoethanol – 1 mL
    • 6 ng/mL bFGF
    Freezing Media
    • 60% FBS
    • 20% hESC media
    • 20% DMSO
    Fixing PSCs:
    1. Observe cells in 6-well dish under microscope and choose best sample for flow analysis. One 6-well should be more than enough to obtain desired amount of cells to perform flow cytometery analysis, ~3x106 total cells. Passage or freeze remaining wells
    2. Aspirate human ES media and add 1 ml Accutase (per well). Place in incubator (37°C) for 30 minutes to create single cell solution
    3. Transfer solution to a 1.5 mL microcentrifuge tube and centrifuge 500g for 5 minutes
    4. Resuspend pellet with 500 uL of PBS and centrifuge 500g for 5 minutes
    5. Repeat PBS wash two more times
    6. Resuspend pellet with 300 uL of BD Cytofix fixation buffer and leave at room temperature for 20 minutes
    7. Centrifuge cells for 500g for 5 minutes
    8. Wash with 500 uL of PBS and centrifuge 500g for 5 minutes
    9. Repeat PBS wash one more time.
    Preparing Antibody Master Mix:
    1. Obtain three microcentrifuge tubes per sample. Label each tube with the sample ID and identify each tube as the Stain, Isotype and Unstained samples
    2. Place these tubes to the side
    3. Obtain two microcentrifuge tubes. Label each tube with Master Stain Mix and Master Isotype Mix
      Master Stain Mix should obtain these antibodies from the kit:
      • PE hNanog
      • PerCP-Cy5.5 Oct3/4
      • Alexa Fluor 647 Sox2
      Master Isotype should obtain these antibodies from the kit:
      • PE isotype control
      • PerCP-Cy5.5 isotype control
      • Alexa Fluor 647 isotype control
    4. Aliquot needed volume of each antibody to the corresponding tube; 20 uL of each antibody per sample
      • If you have 5 samples, add 100 uL of each antibody to the corresponding Master Mix tube. The total volume for this example would be 300 uL
    5. Place the Master Mix tubes in the dark at 4°C
    Preparing Control Beads:
    1. Obtain 4 microcentrifuge tubes and label them Negative, PE, PerCP and 647
    2. Dilute the Perm Wash (10X) provided by the kit to a 1X solution with PBS
    3. Transfer 100 uL of 1X Perm Wash to each tube
    4. Add one drop of negative control beads from the kit to the negative tube
    5. Add one drop of anti-mouse beads to the PE, PerCP and 647 labeled tubes
    6. Pipet 20 uL of PE hNanog, PerCP-Cy5.5 Oct3/4 and Alexa Fluor 647 Sox2 antibodies to the corresponding labeled tube. Do nothing to the negative sample
    7. Incubate in the dark at room temperature
    8. Centrifuge 500g for 5 minutes
    9. Wash with 1X Perm Wash and centrifuge twice
    Treating PSCs for Flow Cytometry:
    1. Wash cells with 600 uL of 1X Perm Wash solution, centrifuge at 500g for 5 minutes
    2. Resuspend pellet with 600 uL of 1X Perm Wash solution and incubate at room temperature for 10 minutes.
    3. Divide volume evenly into the three labeled tubes
    4. Centrifuge at 500g for 5 minutes
    5. Resuspend pellets of tubes labeled S and I with 60 uL of corresponding Master Mix antibodies, keep out of light and incubate at room temperature for 30 minutes. Place unstained samples in 4°C until later steps
    6. After incubation, add 200 uL of 1X Perm Wash to each sample and centrifuge 500g for 5 minutes
    7. Wash with 1X Perm Wash and centrifuge at 500g for 5 minutes
    8. Repeat 1X Perm Wash once more with all samples
    9. Resuspend all sample and control bead pellets in 200 uL of PBS and transfer to 5 mL polystyrene round-bottom tube with cell-strainer cap for Flow Cytometery analysis
    This protocol was largely based on the instruction manual provided by BD Biosciences with the purchase of the Human Pluripotent Stem Cell Transcription Factor Analysis Kit - http://www.bdbiosciences.com/ds/pm/others/23-12787.pdf