Trypan blue stain 0.4

U87NS cells were pH dissociated, filtered, and plated at 250,000/10mL in T75 flasks. Cells were treated with DMSO, JLK1486, TMZ, or TMZ+JLK1486. On day 7 cells were given fresh media and dosed a second time with JLK1486. On day 14 cells were pH dissociated and re-plated at 172,000/10mL in T75 flasks. Cells were pH dissociated and positive and negative trypan blue cells (GIBCO, Trypan Blue Stain 0.4%, #15250) were counted.

Quercetin, epigallocatechin gallate (EGCG), poloxamer 407, and hyaluronic acid sodium salt (HA) with Mws of 8000–15,000 were purchased from Myskinrecipe (Bangkok, Thailand). Poly(DL-lactic-co-glycolic acid) (50:50) with a terminal carboxyl group (PLGA, inherent viscosity 0.22 dL/g, molecular weight of 6700 Da) was purchased from Lactel Absorbable Polymers (Birmingham, AL, USA). MTT was purchased from Roche Applied Science (Penzberg, Germany). Keratinocyte serum-free medium (SFM), insulin, human recombinant zinc solution, collagen I bovine, human fibronectin, Dulbecco’s phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin-streptomycin, trypan blue stain 0.4%, 0.25% trypsin-EDTA, and fluorobrite DMEM were purchased from Gibco (Waltham, MA, USA). The HCE cell line, supplements for keratinocyte consisting of epidermal growth factor (EGF), and bovine pituitary extract (BPE) were purchased from ATCC (Manassas, VA, USA)). Hydrocortisone solution and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma-Aldrich (St. Louis, MO, USA)). ROS/superoxide reagent kit was purchased from Abcam (Waltham, MA, USA).

Kasumi-1 and MOLM-13 cell lines were plated at a density of 5 × 10⁵ cells per mL and exposed to different concentrations (200 μM, 100 μM, 50 μM, 10 μM, 0 μM) of odorants (Cinnamaldehyde and Eugenyl Acetate) diluted in medium RPMI-1640 (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) 0.1% dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA). Cells cultured in a medium with and without DMSO were used as the control. The cells were incubated with Trypan Blue Stain 0.4% (Gibco), and the living cells were counted every 24 h using a Neubauer counting chamber.

iPSC lines edited at the HPRT1 locus and controls were plated at 3 × 10⁴ cells per well in a 6-well culture dish, grown for 2 days without HAT, followed by 2 additional days with or without HAT. Cells were harvested on days 2, 3, and 4 post-plating, and re-suspended in 100 µL of AK02N. An 11 μL aliquot of cell suspension was mixed 1:1 with Trypan Blue Stain 0.4% (Gibco) by gentle pipetting, and 10 μL was applied to each side of a Counting Slide (Bio-Rad). Cell numbers were determined with the TC20 Automated Cell Counter (Bio-Rad).
iPSC lines edited at the APRT locus and controls were plated at 1 × 10⁵ cells per well in a 6-well culture dish in media containing Y-27632 and grown for 2 days. 2, 6-diaminopurine (DAP; Sigma) dissolved in DMSO was added to growth media at a final concentration of 10 µg/mL for an additional 2 days, after which the wells were stained with crystal violet and imaged. Treatment with DMSO was used as a control.

Peripheral blood (10–15 mL) was drawn into a tube containing sodium heparin anticoagulant (BD Biosciences, Oxford, UK). PB-MNCs were purified using either Ficoll–Paque Premium (GE Healthcare, Uppsala, Sweden) or Lymphoprep (Stem Cell Technologies, Oslo, Norway). Viable PB-MNCs were counted by the trypan blue exclusion method (Trypan Blue Stain 0.4%; Gibco, Life Technologies, Carlsbad, CA, USA) [43 (link)].

Cell suspensions were diluted with trypan blue (Gibco Trypan Blue Stain (0.4%) REF –061,15250) either 1:10 (10 μl of cells + 90 μl of trypan blue, dilution factor = 10) or 1:1 (10 μl of cells + 10 μl of of trypan blue, dilution factor = 2), and 10 μl of mixture was pipetted into the chamber of in CYTO C-Chip (inCYTO C-Chip, DHC-N01-5, Neubauer Improved). Cells were counted using a brightfield microscope at 20X magnification. Cells that appeared blue in color were counted as dead cells, whereas cells that were transparent were counted as live cells. Cells were also counted on the edges of the grids, and careful measures were taken to avoid counting debris or red blood cells. Cells were counted in 4 corner quadrants, averaged, and multiplied by 10,000 times dilution factor to get cells/ml count. Two separate researchers counted each slide separately, and cell numbers were averaged across researchers. Equation for determining cell number per ml is as follows: $$\left(\mathrm{Q}1+\mathrm{Q}2+\mathrm{Q}3+\mathrm{Q}4\right)/4\ast 10,000\ast \mathrm{Dilution}\mathrm{Factor}=\mathrm{number}\mathrm{of}\mathrm{cells}\mathrm{per}\mathrm{ml}$$
Every sample and its dilution were separately counted 3–4 times by 2 separate researchers to get technical replicates. Total number of cells, number of live cells and number of dead cells were noted.