Rat pheochromocytoma pc12 cell line

Reagents, if not otherwise stated, were purchased from Sigma-Aldrich (Germany). The PC12 rat pheochromocytoma cell line was obtained from ATCC (USA) or Sigma-Aldrich (Germany). RPMI 1640 medium was from PAA (Austria). Calf and horse sera were from BioChrom (UK). Annexin V-FITC Apoptosis Detection Kit was purchased in Roche Diagn. (Germany). Alexa Fluor 488 and Fluo-4 Calcium Assay kit were from Life Technologies (USA). Protein Assay Kit was from Bio-Rad (USA). Primary antibodies against βIII-tubulin and GAPDH were from Santa Cruz Biotech. (USA).

PC12 rat pheochromocytoma cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). PC12 cells were cultured in Ham's F-12K medium, supplemented with 15% HS, 2.5% FBS. Cells were maintained in the presence of 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air.

All reagents, if not separately mentioned, were purchased from Sigma-Aldrich. The PC12 rat pheochromocytoma cell line was obtained from the American Type Culture Collection (ATCC). Maxima SYBR Green Master Mix, M-MLV Reverse Transcriptase, Trizol®, Alexa Fluor 488, Lipofectamine LTX reagent, B27, and Neurobasal were from Thermo Fisher Scientific. Protein Assay Kit was from Bio-Rad. Anti-GAPDH (Cat. No. sc-32233) and anti-histone H3 (Cat. No. sc-517576) were from Santa Cruz Biotechnology. HMGB1 antibody (Cat. No. 3935) was from Cell Signaling Technology. LDH Cytotoxicity Assay Kit was from Cayman Chemical. GcAMP3 calcium sensor was a gift from Loren Looger (Addgene plasmid #22692). Primers were synthesized in the Institute of Biochemistry and Biophysics (Poland).

Rat pheochromocytoma (PC12) cell line (American Type Culture Collection, Manassas, VA, USA) was maintained in antibiotic-free DMEM supplemented with 10% horse serum and 5% FBS at 37°C in a humidified incubator containing 5% CO₂. Primary cortical neurons were isolated from fetal mice at 16-18 days of gestation and cultured as described previously [35 (link)].

Rat pheochromocytoma PC12 cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 6% fetal bovine serum, and 6% horse serum at 37°C in a humidified 7.5% CO₂ incubator. Fresh medium was supplied every other day. Reagents for cell cultures were purchased from Invitrogen Technologies (Carlsbad, CA, USA).

Rat pheochromocytoma PC12 cell line was obtained from American Type Culture Collection (Rockville, MD) and maintained as a suspension culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 5% (v/v) FBS, 10% (v/v) HS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Cells were grown in a humidified atmosphere containing 5% CO₂ in air at 37 °C in an incubator. For sub-culturing, loosely attached cells in the flask were collected and centrifuged at 304 g for 4 min at room temperature. The cell pellet was washed with HBSS under sterile conditions, re-suspended in 1 ml complete medium and passed through 25 gauge needle 4–5 times to obtain singlet cells. An aliquot of it was used for cell counting with 0.4% trypan blue on hemocytometer under a light microscope. Dye stained cells (blue) were counted as dead cells, while dye excluded cells were counted as viable. The actual cell numbers were determined by multiplying diluted times compared with initial cell numbers.