Roswell park memorial institute (rpmi)

Murine melanoma B2905, derived from a UV-irradiated HGF-transgenic mouse on a C57BL/6 background (Patel et al., 2017 (link)) and B16F10.9 (Porgador et al., 1991 (link)) were used. The B2905 cell line was grown in RPMI (Biological Industries) containing 10% heat inactivated FBS (GIBCO), 1% L-glutamine (Biological Industries), 1% Penicillin/Streptomycin antibiotics (Invitrogen) and 12.5mM HEPES (Sigma) buffer. B16F10.9 cells were grown in DMEM medium (GIBCO) containing 10% heat inactivated FBS, 1% L-glutamine, 1% Penicillin/Streptomycin antibiotics. Both cell lines originated from male mice. Primary cell lines that were generated from solid tumors, derived from UVB-B2905 as described below, were grown in Tu2% media [80% MCDB 153 (Sigma), 20% Leibovitz’s L-15 (Sigma), 5 μg/mL bovine insulin (Sigma), 2% FBS (GIBCO), and 1.68 mM CaCl₂]. The murine lymphoma mutant cell line RMA-s (Ljunggren et al., 1990 (link)) were cultured in RPMI medium supplemented with 10% heat-inactivated FBS, 40 μg/ml gentamycin sulfate and 5x10⁻⁵M β-mercaptoethanol. All cells were cultured using standard procedures in a 37°C humidified incubator with 5% CO₂. Cells were tested routinely for Mycoplasma using Mycoplasma EZ-PCR test kit (cat#20-700-20, Biological Industries).

HEK 293 T cells (ATCC, CRL-3216), HEK 293A GFP-LC3 stable cell line (a kind gift from Sharon Tooze, The Francis Crick Institute, London, England [47 (link)]), PANC-1 cells (ATCC, CRL-1469) and RAW 264.7 cells (ATCC, TIB-71) were grown in Dulbecco Modified Eagle Medium (DMEM; Biological Industries, 01–055-1A). NCI-H1299 cells (ATCC, CRL-5803) and NCI-H460 (ATCC, HTB-177) were grown in RPMI (Biological Industries, 01–104-1A), and HEC-1A (ATCC, HTB-112) in DMEM/F12 (Thermo Fisher Scientific-Gibco, 21,331–020) medium. THP-1 cells (ATCC, TIB-202) were grown in suspension in flasks in RPMI medium. All media were supplemented with 10% fetal bovine serum (Thermo Fisher Scientific-Gibco, 12,657–029), 1% penicillin-streptomycin (Biological Industries, 03–031-1B), 1% glutamine (Biological Industries, 03–020-1A). Cells were routinely monitored for mycoplasma contamination.

To obtain cardiac macrophages, hearts were harvested 4 days after MI, and total heart cells were isolated using an enzymatic digestion mixture,31 which was added to the heart for 3 15‐minute cycles in a rotating water bath warmed to 37°C. Macrophages were purified based on plastic adherence, as previously described.32 Briefly, cells were incubated for 2 hours at 37°C in humid air with 5% CO₂ on 6‐well plates supplemented with RPMI (Biological Industries, Beit HaEmek, Israel) with 10% FBS and 1% penicillin‐streptomycin (Biological Industries, Beit HaEmek, Israel). Then, nonadherent cells were washed, and, to further ensure macrophage enrichment, a 3‐minute trypsin‐EDTA treatment was added to the remaining cells.3 The trypsin was then blocked with fresh medium and washed, and the intact adherent cells were considered cardiac macrophages. To determine purification efficiency, the cells were stained with the macrophage marker F4/80 and displayed >90% positive staining for F4/80 in culture. Next, cardiac macrophages were either immediately lysed for RNA purification or grown for 24 hours for conditioned media collection.

NK92 cells were a kind
gift from Prof. Angel
Porgador from the Faculty of Health Sciences at Ben-Gurion University.
The melanoma cell line 888 was cultured in DMEM and the K562 cell
line was cultured in RPMI (Biological Industries, Beth Haemek, Israel),
supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological
Industries, Beth Haemek, Israel), and maintained in a 5% CO₂ incubator at 37 °C. NK92 cells were cultured in α-MEM
medium (Biological Industries, Beth Haemek, Israel) supplemented with
15% FBS, 15% horse serum, 10% myo-inositol (Biological Industries,
Beth Haemek, Israel), 10% MEM sodium pyruvate (Biological Industries,
Beth Haemek, Israel), and 200 IU/mL IL-2 and maintained at 37 °C
in a 5% CO₂ incubator.