Table 1:

Common live cell stains for imaging platelets in vitro under flow conditions. Vendor list is not complete and not intended as an endorsement, but instead is a suggested starting point to help the reader. Unlabeled primary antibodies may also be pre-labeled with fluorophore conjugation kits.

Purpose	Dye	Concentrations	Vendors	Incubation time/temperature	Useful Tips
Platelet-specific staining	CD41 primary antibody (clone M148) + Any color tagged Secondary Antibody (I.e Goat anti-mouse IgG)	CD41: 1:400 Secondary Antibody: 1:400	CD 41: Novus Biologicals Secondary Antibody: ThermoFisher	Pre-incubation not necessary	Mix primary and secondary together immediately before the experiment
Membrane stain (non-specific)	CellMask (Orange, Deep Red, or Green)	1:1000	ThermoFisher	Pre-incubation not necessary	For prolonged live studies, minimize dye concentration and fluorescent imaging exposure times
Cytosolic calcium	Oregon Green BAPTA-1, AM + Fura Red, AM [66]	BAPTA-1: 1uM, Fura: 1.25 uM	Molecular Probes, ThermoFisher	Incubate for 30 min at 37C	Can be used as ratio for more precision, or used independently
Mitochondrial Staining (non-specific)	MitoTracker Red CMXRos MitoTracker Green FM	100nM [67]	ThermoFisher	Incubate for 15 min at 37C	Keep dye concentration low, high concentrations stain other cellular structures
Platelet activation (phosphatidyl serine)	Annexin V 488 [68]	1:20	Sigma; ThermoFisher	Pre-incubation not necessary, 5 mins sufficient if not staining in real-time	Conjugate fluorophores ranging from AF 250 to AF 680 available
Platelet activation (P-Selectin)	AK4, SC-19996 Ab + Any color tagged Secondary Antibody	1:300 [3]	BD Biosciences, Santa Cruz Biotechnology	AK4: 1 hr incubation Secondary Antibody: 1 hr incubation	May block function, but can be used in low conc. to minimally impact agg [5]
Platelet activation (α_IIbβ₃)	PAC-1 Ab (human) [69] JON/A (murine) [70]	In conjunction with secondary (1μg/mL) [66], Conjugated with fluorophore (PAC1 FITC) (40μg/ml) [71]	Unconjugated: Invitrogen, ThermoFisher Conjugated: BD Biosciences (25ug/mL, conjugated)	5 min sufficient if not staining in real-time [3]	Competitively binds, can reduce adhesion.