Dioc6

Cells were incubated with 10 nM DiOC6 (Enzo) or 5 μg/ml JC-1 dye (Invitrogen) in culture medium for 30 min at 37 °C, and then washed with PBS. For JC-1 staining, the ratio of the mean fluorescence intensity for the phycoerythrin channel to the FITC channel was calculated to correct for variations in the level of JC-1 uptake and cell size. This ratio was expressed as a fold change with respect to CL subset.

HSA surface expression was detected using APC anti-mCD24 (M1/69; BD Pharmingen). Annexin V (Invitrogen) staining was performed per manufacturer’s instructions with or without the presence of 7AAD (10 ug/mL; BD Pharmingen). Intracellular p24Gag staining was performed with phycoerythrin (PE)-conjugated HIV-1 p24Gag antibody (KC57-RD1; Beckman Coulter) with the a Cytofix/Cytoperm kit (BD Bioscience). DIOC6 (Enzo) staining was performed per manufacturer’s instructions. Briefly, cells were resuspended with 2 μM DIOC6 in serum free RPMI and incubated for 20 min at 37 °C, then washed three times with pre-warmed serum supplemented RPMI.
Flow cytometry was performed on a Becton–Dickinson FACSort flow cytometer upgraded with 3 lasers by Cytek Development, Fremont, CA, as previously described [18 (link)]. Compensation was applied during data collection on the basis of single-color controls. Flow data were analyzed with FlowJo software (version 9; Tree Star). Cell sorting was performed using a BD FACSAria at the New York University Langone Cytometry and Cell Sorting Facility.

Analysis of thrombus formation was performed using an Ibidi® μ-Slide VI^0.1 flow chamber (Thistle Scientific Ltd, Glasgow, UK) connected to a Harvard Apparatus syringe pump (Kent, UK). Prior to whole blood perfusion, the Ibidi μ-slide channels were coated with 50 μg/mL^− 1 fibrillar collagen for 2 h at room temperature and blocked in 2% fatty acid free BSA (Sigma) overnight at 4 °C. Channels were flushed with modified HEPES-Tyrodes immediately prior to whole blood perfusion. Anti-coagulated blood drawn in 40 μM PPACK and 2 U mL^− 1 heparin was loaded with 2 μM DiOC6 (Enzo Life Sciences) in the presence of vehicle control or 100 nM AMD3100 for 10 min. Labelled blood was then perfused at a constant shear rate of 1000 s^− 1 for 6 min, followed by a 4 min wash/fixation step in modified HEPES-Tyrodes containing 4% para-formaldehyde at a similar shear rate. Images of platelet thrombi were captured through a 40× oil immersion objective on Leica DM IRE2 inverted epifluorescent microscope attached to a Leica TCS-SP2-AOBS confocal LSM. Five randomly chosen fields of view were analysed per sample with a z-axis interval of 1 μm per confocal section. Quantification of surface coverage was performed with ImageJ and thrombus volume using Volocity® 6.1.1 Quantitation software (Perkin Elmer Inc., San Jose, CA, USA).