Facsaria 2 sorter

For RGC purification, dissected retinas were digested in papain, and dissociated to single cells by gentle pipetting. Retinal cell suspensions were washed in HBSS once, resuspended in HBSS+4% BSA and incubated with PE-Cyanine7 conjugated CD90.2 (Thy-1.2) Antibody (1:2,000, Thermo Fisher Scientific, 25–0902-81) for 15 min to label RGCs for cell sorting (Lu et al., 2020). After another wash with excess HBSS and resuspension in HBSS+4% BSA, DAPI (1mg/ml, 1:1000, Thermo Fisher Scientific, 62248) was added before filtering to label dead cells. Fluorescence-activated cell sorting (FACS) was performed with a BD FACSAria II sorter (BD Biosciences) to collect RGCs.
For immunoblotting, purified RGCs were lysed by heating at 95 ℃ in Laemmli Sample Buffer. Proteins were separated by SDS–PAGE and electro-transferred onto PVDF membranes. Antibodies: pCaMKII (1:1000, Abcam, ab32678), WesternSure Goat anti-Rabbit HRP (1:50000, LI-COR Biosciences, 926–80011), Recombinant HRP Anti-GAPDH (1:400000, Abcam, ab201822). SuperSignal™ West Atto Ultimate Sensitivity Substrate (Thermo Fisher Scientific, A38555) and ChemiDoc Touch Imaging System (Bio-Rad) were used for chemiluminescence detection. The pooled lysate of purified RGCs was used to run 3 independent blots. Images were analyzed using ImageJ (Schindelin et al., 2012 (link)) and Photoshop.

We performed FACS analysis and sorting of THY1⁺PDGFRα⁺ and THY1⁻PDGFRα⁺ cells from primary fibroblast cultures at passage 3. FACS analysis was performed on an LSR II flow cytometer (BD Biosciences), and FACS sorting was performed on a BD FACS Aria II sorter, using a 100 μm nozzle. FACS data were analysed using FlowJo v.10.0.7. Gating was determined using fluorescence-minus-one controls for each colour used in each FACS experiment to ensure that positive populations were solely associated with the antibody for that specific marker (Extended Data Fig. 10). For FACS analysis of cultured cells, fibroblasts were stained with phycoerythrin-conjugated CD140a (BioLegend, 135905) and FITC (fluorescein isothiocyanate)-conjugated CD90.2 (BioLegend, 105305).
EdU incorporation in fibroblast cultures was assessed by FACS using the Click-iT EdU Plus FACS PacBlue Kit (Invitrogen, C10636) in accordance with the manufacturer’s instructions. In brief, fibroblasts were incubated in medium containing EdU (10 μM) for 4 h. Cells were then dissociated and resuspended in FACS buffer (1% BSA in PBS). Cell surface markers were stained with phycoerythrin-conjugated CD140a (BioLegend, 135905) and FITC-conjugated CD90.2 (BioLegend, 105305). Cells were then fixed (4% paraformaldehyde in PBS) and permeabilized, followed by click reaction to detect EdU, according to the manufacturer’s instructions.

Single-cell suspensions were prepared from pancreas as previously described (27 (link)). For isolation of tumor-infiltrating lymphocytes, tumor tissue was minced into 1 to 2 mm pieces and digested with collagenase IV (1.25 mg/mL, Worthington) and 0.1% trypsin inhibitor from soybean (Sigma), in complete RPMI for 25 minutes at 37°C. For isolation and FACS analysis of epithelial and fibroblast compartments, minced tumor tissue was digested with Pronase (0.2 mg/mL, Roche), Collagenase P (0.5 mg/ml, Roche) and DNase I (0.5 mg/mL, Roche). Cells were suspended in 1%FBS/PBS, passed through a 70μm strainer and treated with RBC lysis buffer (eBiosciences). Single cell suspensions were blocked with anti-CD16/CD32 antibody (Fc Block, BD Biosciences) for 5 minutes on ice, and labeled with monoclonal antibodies against mouse antigens as detailed in Supplementary Data. All samples were acquired on LSR II (BD Bioscience) at NYU Flow Cytometry Core Facility and analyzed by FlowJo version 10.2 (TreeStar, Inc.). Cell sorting using a BD FACS ARIA II sorter was performed to isolate Ep-CAM⁺ cells, CD140a⁺ fibroblasts and CD45⁺ cells, and >95% purity of sorted cells was achieved.