Rm1112

The human breast cancer MDA-MB-231 (metastatic) and MCF-7 (non-metastatic) cell lines, and cervical cancer SiHa cell line were procured from NCCS cell repository, Pune, India. J774A cell (Macrophage cell line) was obtained from Dr. Vijay Kumar Prajapati, Department of Biochemistry, Central University of Rajasthan, India. All cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (RM1112, Himedia) and maintained at 37°C in a humidified incubator with 5% CO₂.

Whole blood (8-12 ml) was collected in heparin vacutainers (BD India Pvt. Ltd., Cat#367874) from study participants and PBMCs were isolated by adopting the protocol from Fuss IJ et al., 2009 (32 (link)). Briefly, whole blood was centrifuged at 2600 rpm for 10 minutes to collect the plasma portion. The packed cell volume was diluted with 1X phosphate buffered saline (PBS) (Lonza India Pvt. Ltd. Cat# 17-515F) as per the volume of separated plasma and the initial volume of blood. The PBS-blood mixture was layered on top of the Lymphoprep (STEMCELL Technologies Cat# 07851) gradient solution in 1:1 ratio and centrifuged at 2000 rpm for 30 minutes at 18-20°C without a break to separate the components of blood. The upper layer containing 1X PBS and the remaining cell platelet fraction were removed using the sterile pipet. The mononuclear cells presented as white cloudy layer in between the PBS and Lymphoprep solution was then transferred to fresh tube for further washing procedure. The buffy coat was washed twice with 1X PBS at 1500 rpm for 10 minutes. The final PBMC pellet was suspended with PBS buffer containing 1mM ethylenediaminetetraacetic acid (EDTA) (Himedia Cat# GRM3915) and 2% fetal bovine serum (FBS) (Himedia Cat# RM1112) and the viability of the isolated cells were determined by the trypan blue dye exclusion method.

B16-OVA melanoma cells were kind gift from Dr. Amit Awasthi (TSHTI, India) and were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium (AT162, Himedia, India) with 10% heat-inactivated fetal bovine serum (FBS; RM1112, Himedia, India), 100 U/ml penicillin and 100 μg/ml streptomycin (A002, Himedia, India) and 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; MB016, Himedia, India). The LentiX lentiviral packaging cell line were kind gift from Dr. Rahul Purwar (IITB, India) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; AT007, Himedia, India) containing 10% FBS, 2 mmol/L glutamate (TCL030, Himedia, India), 1% non-essential amino acid (ACL006, Himedia, India), 100 U/ml penicillin and 100 μg/ml streptomycin (Himedia, India). Mice splenic T-cells were cultured in complete RPMI 1640 medium (Himedia, India) with 10% heat-inactivated FBS (Himedia, India), 2 mmol/L glutamate (Himedia, India), 1% non-essential amino acid (Himedia, India), 50 U/ml recombinant human interleukin-2 (rhIL-2; 200-02, Peprotech, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Himedia, India).