Penicillin streptomycin

F11 and CHO K1 cells were cultured in F12 Ham’s medium (HiMedia AL025) supplemented with 10% FBS (HiMedia RM9970), 2 mM l-glutamine (HiMedia TCL012), penicillin–streptomycin (HiMedia A018) (100 units/ml), amphotericin-B (Sigma A2942) 2.5 ng/µl. HaCaT, HEK 293, SaOS and Neuro2a cells were cultured in DMEM (HiMedia) supplemented with 10% FBS (HiMedia, RM9970), 2 mM l-glutamine (HiMedia), penicillin–streptomycin (HiMedia), (100 units/ml), amphotericin-B (Sigma) 2.5 ng/µl. Cells were maintained in a humidified atmosphere at 5% CO₂ and 37°C. For transient transfection, Lipofectamine 2000 (11668027) and Lipofectamine 3000 (L3000015) (Invitrogen) were used according to the manufacturer’s instructions. For transient expression of TRPV2, fluorescent protein-tagged, plasmid-encoding TRPV2-Wt-GFP, mutant TRPV2-N571T, TRPV2-N572T, TRPV2-NN571-572TT were in pcDNA3.1, and actin-RFP were used [22 (link),33 ]. Approximately 30 h after transfection, the cells were fixed by PFA (Sigma). GFP-only was expressed by transfecting the cells with pEGFPN3 vector.

MDA-MB-231 and SKBR3 cells were maintained in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 0.1% penicillin-streptomycin (Hi-Media, Mumbai, Maharashtra, India), 0.1% amphotericin (Hi-Media, Mumbai, Maharashtra, India), and 0.1% ciprofloxacin (Ranbaxy Laboratories Ltd., Gurugram, Haryana, India) in humidified incubator with 5% CO₂ at 37 °C. MCF7 cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin (Hi-Media, Mumbai, Maharashtra, India) in a humidified incubator with 5% CO₂ at 37 °C. Cell lines used in the study were tested for absence of Mycoplasma contamination and validated by morphological observation. All the experiments were performed post 24 h of cell seeding unless otherwise stated.

The human colorectal carcinoma cell line, HCT-116 cells, transfected with pLVX-ZsGreen1-N1 (λ_ex 493 nm, λ_em 505 nm) viral vector were purchased from Clontech (San Jose, CA, USA), as previously reported [12 (link)], and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Himedia, Einhausen, Germany) supplemented with 10% Fetal Bovine Serum (FBS) (Merck-Sigma Aldrich, Milan, Italy), 200 mM L-Glutamine (Himedia, Einhausen, Germany), and 100 IU mL⁻¹ Streptomycin/Penicillin (Himedia Einhausen, Germany), incubated at 37 °C in 5% CO₂. Primary normal human dermal fibroblasts (NFs) extracted from healthy biopsies were grown in Eagle’s Minimum Essential Medium (EMEM, Himedia, Einhausen, Germany), supplemented with 20% FBS (Merck-Sigma Aldrich, Milan, Italy), 200 mM L-Glutamine (Sigma Aldrich), 100 IU mL⁻¹ Streptomycin/Penicillin (Himedia, Einhausen, Germany), 100X Non-Essential Amino Acid (Euroclone, Milan, Italy) and incubated at 37 °C in 5% CO₂.