Cd34 fitc antibody

H9‐derived EBs or CMs were dissociated into single cells by incubation with 0.25% Trypsin‐EDTA (Invitrogen) at 37 °C and fixed with 1% (vol/vol) paraformaldehyde for 15 min at room temperature. For human CD34 staining, cells were incubated with CD34‐FITC antibody (eBioscience) diluted in 100 µL per sample FACS buffer (PBS without Ca²⁺/Mg²⁺, 1% BSA) for 40 min at room temperature and washed twice with FACS buffer before analysis. CMs were incubated with cTnT antibody (Abcam, ab45932) diluted in FACS buffer containing 0.1% Triton X‐100 for 40 min at room temperature, washed twice with FACS buffer, centrifuged, and incubated with secondary antibody in dark for 20 min at room temperature. Cells were washed twice with FACS buffer, centrifuged, supernatant discarded, and resuspended in 400 µL FACS buffer for analysis. Data were collected on flow cytometer (Beckman Cytoflex) and analyzed by FlowJo V10.

Mouse keratinocytes and HFSCs were isolated from the mouse dorsal skin as described previously (Xiong et al., 2022b (link)). Briefly, isolated dorsal skin samples were trypsinized, the epidermis was scrapped, minced, and filtered through a 70 µm cell strainer to prepare primary keratinocytes single-cell suspension. To isolate HFSC, the single-cell suspension was incubated with CD34-FITC antibody (eBioscience, 11-0341-82), Alexa 647-conjugated CD49f antibody (BD Biosciences, 562494), 7-AAD (BD Biosciences, 559925), and different fluorescence minus one was used as a control. Cells were then sorted by BD FACS Aria and analyzed by Flow Jo. All the primary cells were used within 48 hr for experiments.
Human HFDPCs, mycoplasma tested, were purchased from Cell Applications, Inc (cat.# 602-05a). Human HFSCs, mycoplasma tested, were purchased from Celprogen (cat.# 36007-08). Human epidermal keratinocytes, neonatal, pooled, mycoplasma tested, were purchased from Lonza Reagents (cat.# 192906).