Nature research paper: γδ T cells are effectors of immunotherapy in cancers with HLA class I defects
of collagenase II , 10 μg mlof hyaluronidase type IV and 10 μM Y-27632 . Cells were embedded in Cultrex RGF BME type 2 and placed into a 37 °C incubator for 20 min. Human CRC organoid medium is composed of Ad-DF+++ supplemented with 2 mM Ultraglutamine I , 10 mM HEPES , 100 U mlof each penicillin and streptomycin , 10% noggin-conditioned medium, 20% R-spondin1-conditioned medium, 1× B27 supplement without vitamin A , 1.
For organoid surface staining, tumour organoids were dissociated into single cells using TrypLE Express , washed twice in cold FACS buffer , and stained with anti-HLA-A/B/C-PE , anti-B2M-FITC , anti-PD-L1-APC and 1:2,000 near-infrared viability dye , or isotype controls mouse IgG1 kappa . For NKG2D ligand expression analysis, cells were stained with anti-MICA/MICB , anti-ULBP1 , anti-ULBP2/5/6 , anti-ULBP3 , anti-ULBP4 and 1:2,000 near-infrared viability dye .
Analysis was performed using the IncuCyte software for each cancer cell line separately. The following analysis definitions were applied: a minimum phase area of 200 μm, RCU of 2.0, and a GCU of 2.0 and 4.0 . Cancer cell apoptosis was then quantified in the IncuCyte software by counting the total number of green + red objects per image normalized to the total number of red objects per image after 12 h co-culture and displayed as a percentage of two wells with two images per well.
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