Anti human cd34 apc

CD34⁺ CB cells (1 × 10⁶ cells/ml) were labeled with 5 μl carboxyfluorescein diacetate succinimidyl ester (Biolegend, Cat: 423801) according to the manufacture’s guidelines. Labeled cells were cultured for 24 h before they were stained with APC-labeled anti-human CD34 antibody (Biolegend, Cat: 343608) and sorted for CD34⁺CFSE⁺ cells using a FACS MoFlo (Beckman). Sorted cells were then resuspended in HSC expansion medium supplemented with DMSO (0.01%) or JNK-IN-8 (2 µM). Cell aliquots after 2 and 4 days in culture were stained with APC anti-human CD34 (Biolegend, Cat: 343608) and APC-Cy7 anti-human CD45RA antibody (Biolegend, Cat: 304128) before cells were analyzed for CFSE intensity by FCS express version 6 software.

Human CD34+ HSPCs mobilized peripheral blood purchased from AllCells (Alameda, CA, USA) and thawed per manufacturer’s instructions. CD34+ HSPCs were purified from umbilical cord blood collected donated under informed consent via the Binns Program for Cord Blood Research at Stanford University and used without freezing. In brief, mononuclear cells were isolated by density gradient centrifugation using Ficoll Paque Plus. Following two platelet washes, HSPCs were labeled and positively selected using the CD34+ Microbead Kit Ultrapure (Miltenyi Biotec, San Diego, CA, USA) per manufacturer’s protocol. Enriched cells were stained with APC anti-human CD34 (Clone 561; Biolegend, San Jose, CA, USA) and sample purity was assessed on an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Cells were cultured at 37 °C, 5% CO₂, and 5% O₂ for 48 hours prior to gene editing. Culture media consisted of StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml), UM171 (35 nM), and StemRegenin1 (0.75 µM).