Tc20 automatic cell counter

Human normal skin fibroblasts BJ (CRL-2522, ATCC) and squamous carcinoma cells SCC-15 (CRL-1623, ATCC) were cultured in EMEM and DMEM/F-12, respectively. Media were supplemented with 10% FBS and 100 U/mL penicillin/100 μg/mL streptomycin solution, and hydrocortisone (400 ng/mL) for carcinoma. Cells were cultured at 37 °C in humidified 95% air with 5% CO₂, with changing media every 2–3 days and passaged at 70–80% confluence after trypsinization (0.25% trypsin-EDTA in PBS, calcium and magnesium free). The number and viability of cells were estimated by trypan blue exclusion test using Automatic Cell Counter TC20TM (Bio-Rad Laboratories, Hercules, CA, USA). All assays were performed as triplicates of three independent experiments. Cell morphology was observed under Nikon TE2000S Inverted Microscope (Tokyo, Japan) with phase contrast after treatment.

Human immortalized keratinocytes (HaCaT) obtained from Cell Lines Service (Eppelheim, Germany) and human glioblastoma cell line (U-118 MG) obtained from ATCC (Manassas, VA, USA) were cultured in DMEM (doubling time 24 and 35 h, respectively). Normal fibroblasts BJ purchased from ATCC (doubling time 1.9 days) were grown in EMEM medium. Media were supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C in humidified 95% air with 5% CO₂. Media were changed every 2–3 days and cells passaging at 70–80% confluence after treatment with 0.25% trypsin-EDTA/PBS (calcium and magnesium free). Cell morphology was checked using a Nikon TE2000S Inverted Microscope (Tokyo, Japan) with phase contrast. Number and viability of cells were estimated by trypan blue exclusion test using Automatic Cell Counter TC20TM (Bio-Rad Laboratories, Hercules, CA, USA). All assays were performed in triplicate from three independent experiments.

Human glioblastoma cells (U-118 MG, doubling time—37 h) were grown in DMEM, human squamous carcinoma cells (SCC-15, doubling time—48 h) were cultured in DMEM F-12 supplemented with hydrocortisone (400 ng/mL), and normal human skin fibroblasts (BJ, doubling time—1.9 day) were cultured in EMEM. Culture media were supplemented with heat-inactivated 10% FBS and 100 U/mL penicillin and 1% streptomycin solution. All cell lines were cultured at 37 °C in a humidified atmosphere of 95% air with 5% CO₂. Growth media were changed every 2–3 days and cells were passaged at 80–85% confluence with 0.25% trypsin-0.03% EDTA in PBS (calcium and magnesium ions free). Cell morphology was observed with Nikon TE2000S Inverted Microscope (Tokyo, Japan) with phase contrast. Viability and cell density were estimated by trypan blue exclusion test using Automatic Cell Counter TC20TM (BioRad Laboratories, Hercules, CA, USA). All assays were performed in triplicates in three independent experiments. The working solutions of synthesized dendrimer conjugates were prepared from stock solutions in a corresponding cell culture media by adjusting the dmso concentration to 0.05–0.2% (depending on the type of conjugate), which had no effect on treated cells. Control samples with non-treated cells in complete culture medium with adjusted dmso concentration were included in all assays.