Lsrii flow cytometry machine

Tumor cells cultured in vitro were harvested with 0.05% trypsin/EDTA, and washed with PBS three times. Cells were resuspended in media and HS196 or HS199 were added to a final concentration of 10 μM. Cells were incubated in CO₂ incubator at 37C for 30 min, then washed with PBS three times, stained with viability dye (ThermoFisher Scientific, Waltham, MA) and acquired on an LSRII (red laser, APC-Cy7 channel, filter 780/60) (BD Biosciences, San Jose, CA). Data analysis was performed using FlowJo software (FlowJo LLC, Ashland, OR).
For the analysis of tumor cells from in vivo tumors, tumors were minced with a razor blade and digested for 1.5 h at 37°C with triple enzyme buffer, which includes collagenase III (1 mg/ml, Worthington, Lakewood, NJ), hyaluronidase (0.1 mg/ml, Sigma, St. Louis, MO), and deoxyribonuclease (20 U/ml, Sigma). Tumor digests were passed through a 40 μm cell strainer, washed with PBS three times, and acquired on an LSRII flow cytometry machine (BD Biosciences).

Cells were harvested, washed once with PBS and further incubated with the corresponding antibodies in the presence of FACS blocking buffer (1×PBS/10%FCS) for 1 hour on ice in the absence of light. After incubation, cells were washed thrice with 1 ml of FACS blocking buffer and resuspended in a total volume of 200 μl prior to analysis. A minimum of 10,000 cells in the living population were analyzed by using a BD LSRII flow cytometry machine equipped with 5 different lasers and the BD FACSDiva software. Percentages are presented after subtracting isotype background and referring to the total living population analyzed based on FSC/SSC gating and the use of Propidium Iodide and/or 7-AAD staining. Results are representative of at least two independent experiments with a minimum of two technical replicates per experiment.