Alexa fluor488 conjugated anti mouse h2 kd

Active mouse IFN-gamma was sourced from Abcam (Cat#ab9922) and reconstituted in sterile water, as per the manufacturer’s instructions. Subclones (IE1 and IE2) and the parental 4T1 cell line were grown in a 24-well plate until ~70% confluence was achieved. Cells were then treated with IFN-gamma (100 ng/ml) for 24 h. Cells were stained with Alexa Fluor488 conjugated anti-mouse H2-kD (Biolegend, clone SF1-1.1, cat#116610, 1:200) at a concentration of 1:200 in FACS buffer for 20 min. Cells were washed three times before being stained with DAPI. Data were generated using the BD FCSCanto II flow cytometer with BD FACSDIVA software (v8.0.1). Analysis was carried out using FlowJo (version 10.6.1) and the median fluorescence intensity of live single cells was calculated.

5-Aza-2′-deoxycytidine (5-aza) was sourced from Sigma (cat#A3656) and reconstituted in DMSO according to the manufacturer’s instructions. Subclones (IE1 and IE2) and the parental 4T1 cell line were seeded into a 24-well plate at a density of 8000 cells per well in 4T1 media. Cells were allowed to settle overnight before being treated with 5-aza at 200, 100 or 50 nM for 72 h. 5-aza was removed and cells were cultured in media only for 24 h before being collected for flow analysis. Cells were stained with Alexa Fluor488 conjugated anti-mouse H2-kD (Biolegend, clone SF1-1.1, cat#116610, 1:200) at a concentration of 1:200 in FACS buffer for 20 min. Cells were washed three times after staining before being stained with DAPI. Data were collected using the BD FACSCanto II flow cytometer with BD FACSDIVA software (v8.0.1). The resulting data were analysed using FlowJo (version 10.6.1) and media fluorescence intensity of live, single cells was calculated.

The 4T1 subclones (IE1, IE2, NT1 and NT2), as well as the parental 4T1 bulk population, were revived and passaged three times before being seeded into a 6-well plate at a density of 200,000 cells per well. At ~80% confluence, cells were collected into FACS buffer (DPBS supplemented with 2% FCS and 2% HEPEs) for flow cytometry. Cells were stained with a mastermix of APC conjugated anti-mouse CD274 (Biolegend, clone 10 F.9G2, cat#124311, 1:200) and Alexa Fluor488 conjugated anti-mouse H2-kD (Biolegend, clone SF1-1.1, cat#116610, 1:200) in FACS buffer for 20 min. Cells were washed three times with FACs buffer before being stained with DAPI and run on the BD FACSCanto II flow cytometer, utilising BD FACSDIVA software (v8.0.1). Data were analysed in FlowJo (version 10.6.1) and the median fluorescence intensity of live, single cells was calculated.