Cytotoxicity detection kitplus ldh

LDH-assays were performed using the Cytotoxicity Detection KitPLUS (LDH) (Sigma). For this, Hela229 cells were treated with 10 µM C₆-Cer, ω-N₃-C₆-Cer, α-NH₂-ω-N₃-C₆-ceramide and the controls with 10 µl DMSO for 1 or 24 h in 12-well plates. Additionally, one control sample was treated with 20 µl Lysis Solution for 10 min at 37 °C. Afterwards, 500 µl of the cells supernatant was centrifuged at 14.000 g. 100 µl of the centrifuged supernatant was then transferred to a 96-well plate and incubated with 100 µl of a 1:45 mixture of Catalyst (Diaphorase/NAD + mixture) and Dye-solution (INT and sodium lactate). The reaction was performed for 15 min in the dark and then stopped with 50 µl of the Stop Solution. The light absorbance of the samples was then measured on a TECAN infinite M200 and compared to the DMSO treated (low control) and the DMSO and Lysis Solution treated (max control) control samples.

Nisin Z (2.5% w/w, N5764), Pepsin (P0525000), MTT (M2003), and Cytotoxicity Detection Kit ^PLUS (LDH) were obtained from Sigma Aldrich (Milan, Italy). FITC Annexin-V apoptosis kit I (BD Pharmigen™, BD Biosciences, and San Jose, CA, USA, 556547) and fetal bovine serum (FBS) were purchased of Cegrogen-Biotech (Germany, A0100-3010). Pen-Strep (10,000 Unit/mL penicillin and 10,000 unit/mL streptomycin) and 0.25% trypsin-EDTA were purchased from Bioidea (Tehran, Iran). RPMI was prepared from GIBCO Laboratories (Grand Island, NY, USA, 11530586). Coumarin-6 and Oxaliplatin were kindly gifted by Professor Valizadeh’s laboratory (TUOMS, Tabriz, Iran). All other chemicals were of analytical grade.

Equal numbers of subconfluent MCF7 cells expressing GFP alone, cavin1-GFP, cavin2-GFP, cavin3-GFP, and CAV1-GFP were seeded on coverslips. Twenty-four hours later, cells were subject to UV-C exposure for 2 min without media. Complete medium lacking phenol red was added to the cells that were left at 37°C to recover. LDH release assay was measured in triplicate samples from 50 μl of conditioned media expressing cells using the Cytotoxicity Detection Kit^PLUS (LDH) from Sigma-Aldrich according to the manufacturer's instructions. Post-nuclear supernatant from UV exposure cells was also prepared and subjected to western blot analysis with antibodies to BRCA1 (WB 1:500), GFP (WB 1:3000), and Tubulin (WB 1:5000). For knockdown experiments of cavin3 and BRCA1, after 72 hr of knockdown, cells were left untreated or were further transfected with BRCA1-GFP or cavin3-GFP overnight, respectively, and were then subjected to UV exposure 2 min and a recovery time of 6 hr. LDH release was then measured from the cell supernatant in triplicate as indicated in the respective figure legends.