Anti human cd44 antibody

Organoids were dissociated into single cells following the methods outlined above (see Human Organoid Passaging and Maintenance Culture in EHS Matrix and Enteroid Passaging in HELP Matrices). The cells were centrifuged for 5 min at 500 x g to pellet. The media was then removed from each pellet and the cells were resuspended in FACS buffer (PBS + 1 × 10⁻³m EDTA (Invitrogen) + 2% v/v FBS (Atlanta Bio) + 1% penicillin/streptomycin (Gibco)) supplemented with fluorophore‐conjugated primary antibodies (BioLegend anti‐human CD44 antibody, BioLegend IgG2B isotype control). Antibody staining was performed for 30 min at 4 °C in the dark. Following staining, the cells were washed twice using FACS buffer and resuspended in 200 µL FACS buffer with DAPI (1:10000, BioLegend) to select for live cells. Flow cytometry was performed on a Beckman Coulter CytoFlex analyzer (Stanford Stem Cell Institute FACS Core). To analyze the data, gates were determined using forward and side scatter with height and width used to identify cell doublets. Subsequently, live DAPI‐negative cells were gated for all marker analyses and population frequency calculations.

In the standard culture condition, 5 × 10³ cells were incubated in ultra-low attachment, 6-well plates (Corning) containing DMEM/F12, HEPES (Gibco), which included B-27 (Gibco), and 1% PS solution, without disturbing the plates for 6–11 days. For the second-round experiment, the first-round tumorspheres were trypsinized to generate single cells followed by re-suspension with a syringe with a 31-G needle (BD). For CD24 and/or CD44 neutralization, 1 μg of anti-human CD24 antibody (BioLegend, 311102) and 0.15 μg anti-human CD44 antibody (BioLegend, 338802) were added as treatment into each well. To observe the effect of the MCT1 inhibitor, 20 μM of AZD3965 was added as treatment to each cell line. Spheroids were observed microscopically, and the number and diameter of tumorspheres (per field or total) were analyzed on ImageJ.