Rpmi medium

Lungs were harvested, cut into small pieces and digested as follows: for analysis of hematopoietic cells tissue was treated 2 h at 37 °C with 4 mg/ml collagenase D (Roche, Mannheim, Germany) in PBS Ca⁺² Mg⁺² (Biological Industries, Beit Haemek, Israel); for analysis of parenchymal cells tissue was treated 1 h at 37 °C with 1.7 mg/ml collagenase A, 5 U/ml Dispase II, 0.3 mg/ml DNase I (all Roche, Mannheim, Germany) in RPMI medium (Biological Industries, Beit Haemek, Israel). The tissue was then meshed through a 40 μm cell strainer and red blood cells were lysed with red blood cell lysis buffer (Sigma-Aldrich, Rehovot, Israel). Cell suspensions were stained with the following fluorophore conjugated antibodies: CD45 (clone 30-F11), CD11c (N418), MHC class II (M5/114.15.2), Ly6G (1A8), CD170 (S17007L), CD11b (M1/70), CD19 (6D5), CD31 (390), CD326 (G8.8), Podoplanin (8.1.1). Antibodies were purchased from BioLegend, BD Biosciences or eBioscience. For dead cell exclusion, Aqua Live/Dead cell stain (ThermoFisher) was used. Cells were collected by flow cytometry using LSR-Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (version 10, Tree Star, Ashland, OR, USA).

Adherent cultures of PC3 cell line were maintained in RPMI medium (Biological Industries, Beit-Ha'emek, Israel) supplemented with 10% FCS (Biological Industries) and antibiotics. The cells were incubated in a humidified atmosphere of 5% CO₂ in air at 37 °C. Cells were seeded onto 6-well plates (35 mm; Nunc, Copenhagen, Denmark) at a density of 8×10⁵ cells/well and transfected 24 hours later.
Transfection was performed using Lipofectamin 2000 Transfection Reagent according to manufacturer's instructions (Invitrogen). Complete medium was added 24 hours after transfection, for an additional 24 hours, before subjecting the cells to subsequent analysis.

Met-1 mouse mammary gland carcinoma cells were a gift from Prof. Jeffrey Pollard. Met-1 cells were plated on 100 mm plastic dishes and cultured with DMEM medium supplemented with 10% FCS, 1% penicillin-streptomycin, and 1% sodium-pyruvate (Biological Industries). 4T1 mouse mammary cell lines were obtained from the laboratory of Dr. Zvi Granot. 4T1 cells were plated on 100 mm plastic dishes and cultured with RPMI medium supplemented with 10% FCS, 1% penicillin-streptomycin, and 1% sodium-pyruvate (Biological Industries). Cell lines were not authenticated in our laboratory. All cell lines were routinely tested for mycoplasma using the EZ-PCR-Mycoplasma test kit (Biological Industries; 20-700-20).

Human melanoma SK-MEL-5 and SK-MEL-28 and mouse melanoma B16F10 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). The SK-MEL-5 cells were established from a metastatic site (axillary lymph node) of a patient with malignant melanoma and human melanoma SK-MEL-28 cells were established from the skin tissue of a malignant-melanoma patient. Human oral squamous carcinoma HN4 cell line was kindly provided by Dr. Silvio Gutkind (NIDCR), Bethesda, MD, USA. SK-MEL-5, SK-MEL-28 and HN4 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), whereas B16F10 cells were cultured in RPMI medium (Biological Industries, Beit Haemek, Israel). All cell lines were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, all from Biological Industries. Cells were grown at 37 °C in a 5% CO₂ atmosphere.

Amplification buffer was obtained from Invitrogen (CA, USA); Dextran T-500 was obtained from Pharmacosmos A/S (Denmark); Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Biological Industries (Beit Haemek, Israel); ECL solution was obtained from Amersham Biosciences (Buckinghamshire, UK); EcoRI was obtained from New England Biolabs; enhancer solution was obtained from Invitrogen; EX-CELL^® 293 medium was obtained from SAFC Biosciences; fetal calf serum (FCS) was obtained from Biological Industries (Beit Haemek, Israel); Ficoll 1077 was obtained from Sigma-Aldrich (Israel).
Freund’s complete adjuvant (CFA) was obtained from Sigma-Aldrich or Difco; FuGENE^® 6 was obtained from Roche; methylated bovine serum albumin (mBSA) was obtained from Sigma-Aldrich; NotI was obtained from New England Biolabs; NuPAGE^® Bis-Tris gels were obtained from Invitrogen; the pIRESpuro3 vector was obtained from BD Biosciences Clontech; Protein A-Sepharose^® beads were obtained from Amersham; RPMI medium was obtained from Biological Industries (Beit Haemek, Israel); Taq polymerase (Platinum^® Pfx DNA polymerase) was obtained from Invitrogen; VCAM-1 (human) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA); and all the recombinant human chemokines were ordered from PeproTech, Inc. (Rocky Hill, NJ, USA).

OC cell lines; HTB75 (ATCC, CAOV-3; Adenocarcinoma), HTB161 (ATCC, OVCAR-3; Adenocarcinoma) and HTB76 (ATCC, CAOV-4; Adenocarcinoma) cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (01-055-1A; Biological Industries, Beit HaEmek, Israel) supplemented with 10% fetal bovine serum (FBS) (04-127-1A; Biological Industries), RPMI medium (01-100-1A; Biological Industries), supplemented with 20% FBS, and Leibovitz’s L-15 Medium (01-115-1A; Biological Industries) supplemented with 20% FBS respectively. HaCaT (Keratinocytes, CLS Cell Lines Service GmbH, Eppelheim, Germany; [21 (link)]) skin cells were cultured in DMEM supplemented with 10% FBS. All media were supplemented with 1% Pen-Strep, 1% L-Glutamine and 0.02% plasmocin. Cells were incubated at 37 °C in a humidified atmosphere; HTB75, HTB161 and HaCaT were grown in an environment containing 5% CO₂ and 95% air, while HTB76 cells were grown in air. Induction of the mesenchymal phenotype was done as described in [21 (link)], with modifications: HTB75 and HTB161 were seeded in complete medium 3 days prior to induction. Induction conditions for HTB161 were RPMI medium, including FBS 5% and IL-1β 30 ng/mL, and for HTB75 DMEM medium, including FBS 1%, IL-1β 30 ng/mL and TNFα 100 ng/mL for 48 h. Treatments were applied for 16 h, while maintaining induction conditions.