Moflo astrios high speed sorter

For experiments requiring cell sorting, cells were first stained in PBS with 2% FBS and 1 mM EDTA for 15 min, followed by centrifugation at 500g for 5 min and staining with viability factor in PBS for 15 min. Cells were then centrifuged again, resuspended in PBS with 2% FBS and 1 mM EDTA, and sorted using a MoFlo Astrios High Speed Sorter (Beckman Coulter). Sort cells were collected directly in cRPMI. For single-cell RNAseq analysis, live CD45+ cells were sorted by using Fixable Viability Dye eFluor780 (eBioscience) and CD45 conjugated to PE (Biolegend, clone HI30).

TNFR1, TNFR2 and CD271 (NGFR p75) gene knockout plasmids and transfection reagents (UltraCruz Transfection Reagent; Plasmid Transfection Medium; Santa Cruz Biotechnology; Dallas, TX) were used to genetically modify the SK-Mel-37 cell line per manufacturer’s instructions. TNFR2 knockin plasmid (R&D Systems) and transfection reagents (Lipofectamine 3000; Opti-MEM media; ThermoFisher) were used to generate the TNFR2-expressing SK-Mel-28 cell line per manufacturers' instructions. Stable transfectants were enriched by fluorescence activated cell sorting using the MoFlo Astrios High Speed Sorter (Beckman Coulter; Brea, CA).

Healthy murine mammary tissues and EO771 tumors were processed to generate tissue homogenates via mechanical disruption in the presence of Liberase TM (Millipore Sigma) per manufacturer’s protocols. Macrophages from healthy spleen and mammary tissues or EO771 tumors were isolated via flow-sorting sized, LiveDead negative, CD45⁺CD11b⁺F4/80⁺ cells using a MoFlo Astrios High Speed Sorter (Beckman Coulter). Subsequently, cells were lysed in TRIzol reagent (Invitrogen) and RNA was precipitated according to manufacturer recommendations. The isolated RNA was reverse transcribed with SuperScript (Invitrogen). The product of the reverse transcription was then used in triplicates for a qRT-PCR reaction with SYBR Green Fast (ABI Inc.). The CT averages were calculated for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NRP2a, and NRP2b primers as reported previously (8 (link)). Results are reported as the percentage of GAPDH using the formula: %GAPDH = (2−ΔCt) × 100.