Assays performed on day 13 Megakaryocytes. Count cells and resuspend in SFEM without growth factors at 1×10^6/mL . All assays should be done in comparison to a non-targeting crRNA control and if desired a targeting control (i.e. B2M)

Proplatelet formation

  1. coat 8 well borosilicate chamber slide with 100 ug/mL fibrinogen diluted in PBS for 30 min at 37c
  2. remove fibrinogen, block with 2.5% human serum albumin for 20 minutes
  3. wash 3x PBS
  4. add 2.5×10^5 cells in 250 uL SFEM-T per well
  5. incubate overnight
  6. add 100 uL 4% PFA for 20 minutes at RT
  7. Fix with 2% paraformaldehyde
  8. wash 3x with PBS
  9. permeabilize cells with 0.1% triton x 100 for 5 minutes
  10. Wash 3x with PBS
  11. add phalloidin 488 and nuclear stain for 15 minutes at RT
  12. wash 3x with PBS, keep in PBS for imaging
  13. While blinded to treatment, image cells on fluorescent microscope, take ratio of # proplatelet forming vs total cells. Examples of proplatelet forming megakaryocytes are marked with white arrows in the images below.
Picture1-4 Megakaryocyte phenotypic and platelet function assays

Spreading

  1. coat duplicate wells of 96 well plates with collagen (coated the night before) or fibrinogen
    • Dilute collagen 1:10 in HBSS. 40 uL to each well, place at 4c overnight
    • Thaw reconstituted fibrinogen at 37c. Dilute 1:10,000 in HBSS, 40 uL each well place for 30 minutes at 37c
    • Wash wells 3 times with PBS
  2. Add 2.5×10^4 megakaryocytes to wells in 100 uL media.
  3. Incubate 1 hour at 37c
  4. gently remove unbound cells
  5. wash gently with PBS
  6. Fix with 2% paraformaldehyde
  7. process cells for imaging as in steps 8-12 of proplatelet assay
  8. While blinded to treatment, image cells on fluorescent microscope, take ratio of # spread vs round cells. In the example below most of the negative CRISPR treated cells are spread (egg on a pan) on colagen, whereas most GP6 KO cells are round.
Picture1-5 Megakaryocyte phenotypic and platelet function assays

Flow cytometry phenotyping and assays of activation

We do activation assays in a round bottom polypropylene 96 well plate. Include an unstimulated activated and unactivated matching isotype control well for at least one of the conditions.

  1. Prealiquot 50 uL of phenotyping, activation, or fibrinogen binding antibody mix into individual wells. Use SFEM without cytokines:

Phenotyping mix

  • 1 uL CD41-APC
  • 2 uL CD42a-PE
  • 1 uL CD61-FITC
  • 46 uL SFEM

Activation mix

  • 6 uL cd62-PE (granule marker)
  • 10 uL PAC1-FITC (integrin activation)
  • 5 uL CD63-V450 (dense granules/lysosomes, optional)
  • 1 uL CD41-APC (MK marker)
  • 28 uL SFEM

Fibrinogen binding mix

  • 2 uL CD42a-PE
  • 1 uL Fibrinogen-647
  • 47 uL SFEM

2. Add vehicle (for unstimulated) or agonist, such as one of the following, to activation wells:

  • 1 U/mL thrombin*
  • 1.25 ug/mL convulxin
  • 2 ug/mL CRP
  • 200 nM 2-MesSADP

*Thrombin can’t be used in the fibrinogen binding assay without adding inhibitors to prevent clot formation

3. Immediately add 50 uL of cells (5×10^5) and pipet to mix (for many conditions use a multichannel pipetor

4. Incubate at 37c for 15 minutes

5. Fix with 100 uL 4% PFA (2% final)

6. Analyze by flow cytometry

Gate on higher FSC, lower SSC population as described by https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6886406/ . Further gate on singlets using FSC-H and FSC-W. Set phenotype marker (CD41, CD61,CD42a) or activation gates (PAC-1, P-selectin, or CD63) based on isotype controls. Record % positive cells and gMFI of the gated cells. Gating examples shown below for negative control vs ITGA2B KO cells.

last Megakaryocyte phenotypic and platelet function assays