Dulbecco s phosphate buffered saline dpbs without ca2 and mg2

HeLa cervical cancer cells were purchased from Highveld Biological, Johannesburg, South Africa. RPMI 1640 cell culture medium and fetal bovine serum was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS) with Ca²⁺ and Mg²⁺ and Dulbecco’s phosphate buffered saline (DPBS) without Ca²⁺ and Mg²⁺ were purchased from Lonza (Wakersville, MA, USA). Trypan blue, bisBenzamide H 33,342 trihydrochloride (Hoechst 33342), cisplatin, penicillin/streptomycin and bovine serum albumin fraction V were purchased from Sigma-Aldrich (St. Louis, MI, USA). NucRed^TM Live 647, CellRox^® Orange reagent, Lysotracker™ Deep Red and Tetramethylrhodamine ethyl ester (TMRE) were purchased from Molecular Probes^®-Life Technologies-Thermo Fisher Scientific (Logan, UT, USA). Annexin V-FITC/PI kit was purchased from MACS Miltenyi Biotec (Cologne, Germany). Cleaved caspase 3 (Asp175) (D3E9) Rabbit mAb, Cleaved caspase 8 (Asp391) (18C8) Rabbit mAb, Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 647 conjugate), Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 488 conjugate) and Phosphorylated Histone H3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Sigma-Aldrich (Taufkirchen, Germany) was used for chemicals, Biochrome AG (Berlin, Germany) and Greiner Bio-One (Frickenhausen, Germany) as suppliers for cell culture materials, media, and solutions, unless otherwise indicated. Culture medium R10 (RPMI-1640 without l-glutamine, from Lonza, Visp, Switzerland) was supplemented with 10% (v/v) fetal calf serum, 2 mM l-glutamine, and 100 IU/mL penicillin or 100 µg/mL streptomycin prior to use. Culture medium Opti-MEM (reduced serum medium, GlutaMAX™ Supplement, Invitrogen/Life Technology/Thermo Fisher Scientific, Waltham, MA, USA) was used as received from the supplier (Invitrogen/Life Technology/Thermo Fisher Scientific, Waltham, MA, USA). Dulbecco’s Phosphate-Buffered Saline (DPBS) without Ca²⁺ and Mg²⁺ (Lonza, Visp, Switzerland) was used. NaCl (150 mM in Milli-Q water) and HEPES buffered glucose (HBG, 20 mM HEPES, 5 wt % glucose, pH 6.0) solutions were prepared in house and sterilized by filtration (0.2 µm, cellulose acetate filters).

Blood samples were collected in EDTA+ tubes. PBMCs were isolated from whole blood by density gradient centrifugation with Ficoll-sodium diatrizoate (Lymfoprep, Axis-Shield PoC AS, Oslo, Norway) (400 × g, 30 min, 20 °C, without break). As such, PBMCs were separated from the red blood cells and neutrophils according to their density. The top layer contained both PBMCs and plasma and after collecting the PBMCs, they were washed twice with Dulbecco’s phosphate buffered saline (DPBS without Ca²⁺ and Mg²⁺; Lonza, Bazel, Switzerland) and counted in a hemocytometer.