Phycoerythrin conjugated anti mouse cd31

The longitudinal muscle–myenteric plexus layer was harvested from the small and large intestine of adult C57BL/6 mice, minced with scissors, and dissociated enzymatically with dispase (250 μg/mL), collagenase XI (1 mg/mL), and DNase I (1 mg/mL; Sigma Aldrich). Cells were stained with Alexa Flour 647 conjugated anti-mouse CD49b and with the following lineage (Lin) markers: phycoerythrin-conjugated anti-mouse CD31, anti-mouse CD45, and anti-mouse TER-119 antibodies (BioLegend, San Diego, CA). Flow cytometry was performed on an FACS Vantage SE/DiVa SORP (BD Biosciences, San Jose, CA). Purified CD49b⁺/lin⁻ cells were cultured for 5 to 7 days on fibronectin-coated glass chamber slides at 40,000 cells per milliliter in serum-free neural differentiation medium (Neurocult; StemCell Technologies).

Retinas were dissected from freshly collected eyes of P6 neonates and digested with LiberaseTM (Sigma-Aldrich, CAT#5401127001, 0.26 U/mL) and deoxyribonuclease I (Sigma-Aldrich, CAT#D4527, 10 mg/mL) in PBS (1 mL per one retina) at PBS at 37°C for 30 min under constant rotation. At 15 min of digestion period, the suspension was triturated 10 times through a 18G needle to dissociate the clumps. The reaction was stopped by adding BSA (final 1%) and EDTA (final 0.5 mM) and centrifuged at 400 × g at 4°C for 10 min. The pellet was further washed once with FACS buffer (0.5% BSA/0.5 mM EDTA in PBS) and stained with phycoerythrin-conjugated anti-mouse CD31 (1:200, BioLegend, CAT#102508), allophycocyanin (APC)-conjugated anti-mouse CD45 (1:300, BioLegend, CAT#103111) and APC-conjugated TER119 (1:300, BioLegend, CAT#116212) antibodies for 1 hour on ice. The retinal cells were washed twice and treated with DAPI to exclude dead cells. CD31+/CD45-/TER119- cells were sorted using BD FACSAria™ II (BD Biosciences). The cells from 4–6 pooled retinas were sorted in either buffer RLT from RNeasy Micro Kit (Qiagen, CAT#74004) supplemented with β-mercaptoethanol (Sigma-Aldrich, CAT#M3148) or 0.1% BSA/PBS for RNA-seq or ATAC-seq, respectively.
Individual biological replicates for RNA-seq or ATAC-seq represent pooled sorted cells from 4–6 genotype-matched pups (one retina was used for FACS per pup).