Facs fusion flow cytometer

The cell suspensions were prepared by pressing the spleen tissue through a fine nylon mesh followed by 2 washes with 30 mL of cold PBS. After the last washing step, the splenic leukocytes were re-suspended in 1 mL of PBS and used for surface marker staining.
In total 10⁸ of cells were incubated for 20 min with anti-monocyte/macrophage:FITC (clone KUL01 from Southern Biotech) and CD45:APC (clone LT40 from Southern Biotech), followed by wash with PBS. Monocytes/macrophages (CD45+KUL01+) and heterophils (identified based on FSC/SSC characteristics within CD45+ cells) were sorted using a FACSFusion flow cytometer operated by FACSDiva software (BD Biosciences). Only for simplicity, the monocytes/macrophages population will be called as “macrophage (Ma)” in the rest of this paper. Sorted cells were collected in PBS and immediately processed as described below. A small aliquot from each sample was subjected to immediate purity analysis. The purity of macrophages was 88.6 ± 5.3% and of heterophils 88.1 ± 4.2% when counting cell of expected staining, and FSC and SSC parameters out of all particles. When we gated at the area with live cells, the purity of macrophages and heterophils was between 97 and 98%. Majority of contaminants therefore represented cellular debris and only around 2.5% of contaminants were formed by non-target cells.

Cells were analyzed using a Fortessa flow cytometer (BD). Human CD34⁺CD38⁺, CD34⁺CD38^– cells and mouse L^–S^–K^–, L^–S⁺K^–, L^–S^–K⁺, MEP, CMP, GMP, and LSK were isolated by flow cytometry sorting using a FACS Fusion flow cytometer (BD).

Renca cells and 786-O cells were maintained in DMEM containing 10% fetal bovine serum at 37 °C with 5% CO₂ in a humidified incubator. STR DNA fingerprinting of 786-O cells was performed by the CCSG-funded Characterized Cell Line Core, NCI # CA016672. Renca cells were transfected with control knockout CRISPR/Cas9 plasmid (Santa Cruz Biotechnology, sc418922) or murine Pbrm1 CRISPR/Cas9 knockout plasmids (sc-426270) plus homology-directed repair (HDR) plasmid (sc-426270-HDR). Cell transfection were mediated with Lipofectamine 2000 (Invitrogen). HDR Plasmid expresses puromycin resistance gene to enable selection of stable knockout (KO) cells and RFP expression for single-cell sorting. Transfected Renca cells were selected in medium containing 2 µg/ml puromycin, and then single knockout clones were sorted using BD FACS FUSION flow cytometer. 786-O stable cell lines expressing control shRNA or PBRM1 shRNA (Dharmacon, V3LHS_318943 and V2LHS_174972) were infected with lentiviral particles and selected in medium containing 2 μg/ml puromycin.