Ly 6g antibody conjugated to apc cy7

Intestinal tumors were isolated by dissection under a stereomicroscope, minced by scalpels and digested for one hour in DMEM containing 2 mg/ml (560 U/ml) collagenase I (Worthington) and 3 mg/ml Dispase (Roche). Cell suspensions were washed and passed through a 40 μm filter and Fc receptors were blocked using TruStain fcX (anti-mouse CD16/32) antibody (Biolegend cat. no. 101320), then stained with EpCAM antibody conjugated to Brilliant Violet 421 (BioLegend cat. no. 118225), F4/80 antibody conjugated to Alexa Fluor 488 (BioLegend cat. no. 123119), and Ly-6G antibody conjugated to APC-Cy7 (BioLegend cat. no. 127623). Dead cells were stained with propidium iodide (PI) immediately before analysis. Filtered cells were stained with antibodies and sorted on a Moflo Legacy (Cytomation, currently Beckman Coulter) or analyzed on an LSRII instrument (BD Biosciences). For FACS, 100 000–300 000 cells were sorted directly into TRI Reagent (Molecular Research Center). For all analysis, populations were first gated for cells (as opposed to debris) in a FSC-A vs. SSC-A graph, for single cells (as opposed to doublets/aggregates) in a FSC-A vs. FSC-W graph and for live (PI-negative) cells. Compensation was performed using single-stained cells and gating was aided by fluorescence minus one (FMO) controls.