Cd172a b pe

After a 5 min dissociation step with TrypLE™Select at 37 °C, cells were washed with DPBS by centrifugation and a total of 2–3 × 10⁵ cells were used per analysis. For membrane markers, cells were incubated with the primary antibody for 1 h and with the secondary antibody for 30 min at 4 °C in DPBS with 5% (v/v) FBS. For intracellular markers, cells were permeabilized using the Inside Stain Kit (Miltenyi Biotec) according to the manufacturer‘s instructions. The following primary antibodies were used: SirPα/β (1:20, CD172a/b-PE, BioLegend), Troponin T (1:200, TnT, Thermo Scientific), CD105 (1:20,BD Pharmingen), CD166 (1:20,BD Pharmingen), CD44 (1:5, eBiosciences), CD11b (1:10, AbDSerotec), CD34 (1:5, BD Pharmingen), CD45 (1:5, BD Pharmingen), and isotype controls mouse IgG1k (1:5, BD Pharmingen), mouse IgG1 (1:2.5,Santa Cruz Biotechnologies), and rat IgG2b (1:5, eBiosciences). Cells were analyzed in a CyFlow® space (Partec GmbH) instrument, registering at least 10,000 events/sample.

Human iPSC–CM aggregates were harvested from culture and dissociated with TypLE^TM Select for 5 min at 37°C with agitation. Afterward, single cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific), and 5 × 10⁵ cells were incubated in the dark with one of the following conjugated antibodies for 1 h at 4°C: SIRPα/β (CD172a/b-PE, BioLegend, diluted 1:20 in DPBS), VCAM (CD106-PE, BD Biosciences, diluted 1:5 in DPBS), or isotype control immunoglobulin G1 (IgG1),κ-PE (BD Biosciences, diluted 1:5 in DPBS). For detection of intracellular marker (Troponin T, Thermo Fisher Scientific), cells were fixed and permeabilized with Inside Stain Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were incubated with primary antibody (diluted 1:200 in InsidePerm) for 10 min at RT, washed with InsidePerm, and incubated with secondary antibody anti–mouse IgG Alexa Fluor 488 (diluted 1:200 in InsidePerm) for 10 min at RT. Cells were washed with InsidePerm and analyzed by a CyFlow^® space (Partec GmbH). Ten thousand events were analyzed per sample.