Hepes buffer

B16-F10, 4T1, Mel-Rel, TS/A, MCF-7, MDA-MB-468, LAU 145, and Me290 cell lines were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cells were grown until 75% confluency and passaged using trypsin/1× EDTA (Eurobio), washed with PBS, and resuspended in supplemented RPMI-1640.

The murine melanoma B16F10 cell line was kindly provided by PR (Ludwig Center for Cancer Research, Department of Oncology—Faculty of Biology and Medicine University of Lausanne, Switzerland). The B16 mouse model was created in the 1970s from a melanoma that developed spontaneously in the ear of a female C57BL/6 mouse and was then passaged in vivo to create the B16F10 line (21 (link)). The murine breast cancer 4T1 cell line was grown in BALB/c mice and developed into a highly tumorigenic and invasive tumor that can spontaneously metastasize from a primary tumor in the mammary gland to multiple distant sites (22 (link)). The 4T1 mammary tumor cells were kindly provided by Dr Alexzander Asea (Texas A&M Health Science Center College of Medicine, Temple, TX). Cells were cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Eurobio), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M HEPES buffer and 1 mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO₂. Cells were grown until 75% confluency and passaged using trypsin/1X EDTA (Eurobio), washed with PBS and resuspended in supplemented RPMI-1640.

4T1 and B16-F10 cells were cultured in RPMI-1640 medium (Eurobio, Toulouse, France) with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio) and cultivated in a humidified incubator at 37 °C in 5% CO₂.