Lsrfortessa x 20

After tissue disaggregation into a single cell suspension, cells were first stained with a panel of fluorescently labelled antibodies to surface antigens, washed with FACS wash buffer (1% bovine serum albumin (BSA), 0.01% NaN₃ in PBS) then fixed using CytoFix (eBiosciences Foxp3 staining buffer set # 00-5523-00) for a minimum of 30 min at 4 °C. After three washes in perm wash (eBioscience Foxp3 staining buffer), intracellular antigens were stained with another panel of fluorescently labelled antibodies, and cells were then washed with perm wash and analysed by flow cytometry (BD LSR Fortessa X20) and FlowJo software. BD comp beads (anti-mouse Ig k #552843) were used to establish compensation settings. Dead cells were deselected using live/dead stain, added to surface staining panel prior to cell fixation (Life Technologies Live/Dead^TM Near IR fixable dead cell stain kit #L10119). Isotype controls were used during antibody optimisation.

Samples were analysed on a BD LSR Fortessa™ X-20 and data analysed on FlowJo 9.6.6 software.

To determine if colchicine causes GPVI receptor shedding, flow cytometry was performed under non-stimulated conditions. Citrated PRP was incubated with colchicine as indicated above. Where GM6001 was used the PRP was preincubated with 250 µM GM6001 for 15 min at room temperature prior to the colchicine incubation to inhibit metalloproteinase induced shedding. The positive control for GPVI receptor shedding was 5 mM NEM, the incubation was performed concurrently with colchicine under colchicine incubation conditions. After the incubations were performed, PRP was incubated in the presence of GPVI-AF488 antibody, CD61 PerCP-Cy5.5 and DPBS. The samples were then fixed with 0.16% paraformaldehyde solution in saline, run on a BD LSRFortessa X-20 and analysed by FlowJo X.0.7. Positive percentage GPVI expression was determined by gating CD61 positive platelets on the positive peak from the NEM incubated sample. Geometric mean was used for the mean fluorescence intensity (MFI) result.

The FITC Annexin V Apoptosis Detection Kit I was used to measure cell apoptosis. Second passage astrocytic cells were seeded in 6-well plates at 5 × 10⁵ cells/well for each group for 24 hours. Cells were treated with different concentrations of SFN for 24 hours when fully attached. Both floating and adherent cells were collected and washed twice with cold PBS. Then, cells were resuspended in 500 μl binding buffer, 5 μl Annexin V-FITC, and 5 μl propidium iodide (PI) and incubated in the dark at room temperature for 15 minutes. Cell apoptosis was detected by BD LSRFortessa X-20 and analyzed by FlowJo software.

PBMC from newborns d10 p.b. were purified from peripheral blood by density gradient separation, aliquotted and stored in liquid nitrogen. For phenotyping by flow cytometry, cryopreserved cells were thawed in culture media as above and rested at 37 °C for 2 h. Cells were stained with Fixable Viability Stain 780 (BD Horizon). Surface staining was performed using the following antibodies: CD3-PE Violet 770 (clone 10D12), CD4-PerCP-Cy5.5 (clone L200), CD20-AlexaFluor 700 (clone 2H7), CXCR5-PE (clone MU5UBEE), PD-1-PE-Dazzle 594 (clone EH12.2H7), ICOS-BV510 (clone C398.4A). Cells were then washed, fixed and permeabilized with FoxP3/Transcription Factor staining kit, followed by FoxP3-BV421 (clone 206D). Samples were acquired on a BD LSRFortessa X-20 and analyzed with FlowJo software.

p53WT and p53KO hiPSCs were treated with 25 and 50 µg/mL EPS for 24 h, harvested, washed twice with cold D-PBS, and then stained with FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. After labeling with annexin V-FITC and PI, cells were analyzed using an LSRFortessa X-20 and FlowJo software.