Cd326 pe cy7

Wild-type organoids (P1) from n = 16 wells per condition were pooled and collected in Gentle Cell Dissociation Reagent and subsequently dissociated into single cells using the TrypLE Express solution (Gibco) with DNase I (0.1 mg/ml) as described. Single cells were resuspended in FACS buffer (PBS with 2% BSA or FBS), filtered and counted. 10⁵ - 10⁶ cells were resuspended in FACS buffer with 1 μg/ml TruStain FcX™ anti-mouse CD16/32 antibody (BioLegend) to block for 5 minutes before adding conjugated surface antibodies (CD326-PE-Cy7 (1:200) (BioLegend)) in FACS buffer. Cells were stained for 20-30 minutes at 4°C in the dark and washed twice in FACS buffer. Cells were resuspended in FACS buffer with DAPI (33 ng/ml) at a final concentration of 5-6 million cells/ml for subsequent fluorescence-activated cell sorting into a pre-coated tube filled with FACS buffer. Cells were then spun down at 1400 rpm for 5 minutes and resuspended to reach a final concentration of 1200 cells/μl. 1.5 x 10⁵ cells from the organoids only condition and 10⁵ cells from co-cultured organoids were collected.