Recovery cell culture freezing media

Retinal tissue was isolated from donors 5–8 with 1 mm (foveal) and 4 mm (parafoveal) trephine punch biopsies centered on the fovea. To minimize inadvertently collecting cells from the retinal pigment epithelium or choroid, an 8 mm retinal punch was first collected and moved to a separate dish, and the 1 and 4 mm samples were collected from the excised macula before gentle dissociation in 20 units/ml of papain with 0.005% DNase I (Worthington Biochemical Corporation, Lakewood NJ, USA) on a shaker at 37°C for 1–1.25 h. The dissociated retinal samples were resuspended in DMSO-based Recovery Cell Culture Freezing Media (Life Technologies, Grand Island NY, USA) and frozen at −80°C in a CoolCell LX container (Corning NY, USA) to cool at 1°C/min. After samples were frozen for 3–12 h, they were transferred to liquid nitrogen for long-term storage.
Cryopreserved retinal samples were rapidly thawed in parallel at 37°C and resuspended in phosphatidylcholine buffer solution with 0.04% non-acetylated bovine serum albumin (New England Biolabs, Ipswich MA, USA) at a concentration of 1000 cells/μl. Single cells were then microfluidically separated and barcoded with the Chromium system v3.1 chemistry kit (10X Genomics, Pleasanton CA, USA). Resulting libraries were pooled and sequenced on the NovaSeq 6000 platform (Illumina, San Diego CA, USA), generating 100-base pair paired-end reads.

To prepare the primary CEFs, the body walls of 10-day-old chicken embryos were chopped with a knife, trypsinized, and collected by centrifugation^{35 (link)}. Enzymatic activities were stopped with fetal bovine serum (FBS), and the cells were washed once with PBS. The culture media contained 15% FBS (HyClone, Logan, UT), 100 U/ml penicillin (HyClone), and 100 µg/ml streptomycin (HyClone). The harvested cells were plated and maintained at 37 °C with 5% CO₂. The 3D8 scFv transgenic cells isolated from transgenic eggs were selected by 2 µg/ml Puromycin (Sigma, St Louis, MO) for 3 days. The surviving cells were selected and amplified in the same media containing 2 µg/ml Puromycin for subsequent experiments. The amplified cells were frozen in Recovery™ Cell Culture Freezing Media (Gibco, Grand Island, NY, USA) for later experiments.