Tc20 automated cell counter

Day 67 GLAST-positive cells were thawed and seeded into hESC-qualified matrigel coated 6-well plates at 4e4 cells/cm² in ADM3 (see astrocyte differentiation). After 48 hours, day 69 GLAST-positive cells were washed with HBSS without Mg Ca (Gibco) three times then lifted using a 10–15 minutes incubation of StemPro Accutase (Gibco) at 37°C. Single-cell suspensions were strained using a 70 μm strainer, washed using cold 0.08% BSA (Gibco) in PBS, and counted using TC20 Automated Cell Counter (BioRad) with trypan blue exclusion per manufacturer’s instructions in the 10x Genomics® Demonstrated Protocol: Single Cell Suspensions from Cultured Cell Lines for Single Cell RNA Sequencing (Document CG00054). To obtain a single-nuclei suspension, cells were lysed using Nuclei EZ Lysis Buffer (Sigma Aldrich, NUC101-1KT) for 5 minutes on ice. Lysed cells were centrifuged 500 rcf for 5 minutes at 4°C, then washed by twice resuspending in 1% BSA (Gibco), 0.2 U/μL of Human Placenta RNase Inhibitor (NEB), in PBS and centrifuged again. After two washes, the nuclei were strained using a 40 μm cell strainer, counted using the TC20 Automated Cell Counter (Bio-Rad), resuspended at 1000 nuclei/μL in 0.08% BSA (Gibco) in PBS, then proceeded immediately with the 10x Genomics® Chromium Single cell 3’ gene expression v3 platform. Libraries were quality controlled and sequenced on the Illumina NovaSeq 6000.

Dorsal air pouches were prepared by injection of 2.5 mL of air on day 0 and day 3 in CD-1 mice, as previously described.25 (link) On day 6, mice received 0.25 mL of one of the following treatments diluted in 0.5% carboxymethyl cellulose (CMC, Sigma-Aldrich): (1) vehicle, CMC alone; (2) IL-17 (1 µg); (3) nIL-17 (1 µg); (4) IL-17 (1 µg) plus MAB421 or Ab-IPL-IL-17 (10 µg); (5) IL-17 (1 µg) plus anti-JE (10 µg, MAB479, R&D System) and (6) IL-17 (1 µg) plus anti-KC (10 µg, MAB453, R&D System). Mice were sacrificed after 24 hours, and lavage fluids were recovered, and centrifuged at 220 g for 10 min at 4°C. Cell pellets and inflammatory exudates were banked for subsequent analysis of inflammatory cyto-chemokines. The route, timing and frequency of administration as well as the selected dosages of tested compounds were selected according to updated literature.12 16 (link) Cell number was determined by TC20 automated cell counter (Bio-Rad) using Bio-Rad’s TC20 automated cell counter uses disposable slides, TC20 trypan blue dye (0.4% trypan blue dye w/v in 0.81% sodium chloride and 0.06% potassium phosphate dibasic solution, Sigma-Aldrich) and a CCD camera to count cells based on the analyses of capture images.25 (link)

HCEC, LoVo and SW480 cells were treated with pharmacological concentrations of vitamin C (0-10mM) for 2h, and then replaced with normal DMEM supplemented with 10% fetal calf serum (FCS). For time dependent viability assays, LoVo and SW480 cells were treated withvitamin C (10 mM) for 2h and then collected (0, 5, 12, 36 h) for cell counting using a TC20™ Automated Cell Counter (Biorad). For cetuximab and vitamin C combinatorial assays, HT29 harboring wild type KRAS, and the KRAS mutant LoVo and SW480 were treated with cetuximab (calculated IC50=0,4 μM), vitamin C (5mM) alone and combination for 12 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad).

Human PCa cell lines were purchased from the American Type Culture Collection (ATCC, Burlington, ON, Canada). Cells were cultured in RPMI‐1640 media (Gibco, Burlington, ON, Canada, Cat# 11875‐093) supplemented with 10% FBS (Gibco, Cat# 10099‐141) and following ATCC protocols for culture passage and storage of cells (ATCC, cryogenic storage of animal cells protocol). A humidified 37 °C 5% CO₂ incubator was employed for all culturing. Genetic fingerprinting and monthly Mycoplasma tests were conducted at the Vancouver Prostate Centre. Unless otherwise stated, all cells were counted for experiments using a TC20 automated cell counter (Bio‐Rad, Mississauga, ON, Canada, Cat# 1450102) following manufacturer's protocol (TC20 automated cell counter quick guide) and were maintained until a passage of 20 or lower.

To analyze cell proliferation, 5 × 10⁴ DAOY and ONS cells were seeded into 12-well plate, transfected with siCtrl or siCENPE and counted in triplicates after 48, 72 and 96 h with Bio-Rad TC20 Automated Cell Counter. To assess the effects of GSK923295, 5 × 10⁴ DAOY and ONS cells were seeded into 12-well plate, treated with DMSO or 25 nM GSK923295 and counted in triplicates after 24, 48 and 72 h with Bio-Rad TC20 Automated Cell Counter.

In vitro studies with CAR-T cells (CD19-CD28z and CD19-BBz) were performed for the assessment of efficacy and cytotoxic capacity. For that purpose, anti-CD19-expressing CAR-T cells and CD19-expressing RAJI cells were cultured for 24 h, 7 days, and 14 days (CAR-T: RAJI; 1:1, 5:1, 10:1). Anti-cancer profiling studies on days 14 and 21 were analyzed using CD3-PC7 (Beckman Coulter, 6607100), CD4-APC-A700 (Beckman Coulter, B10824), CD8-PC5.5 (Beckman Coulter, B21205), EGFR-A488 (R&D Systems, FAB10951G), CD19-ECD (Beckman Coulter, A07770), CD25-APC (Miltenyi Biotech, 130-113-284), and CD107a (LAMP-1) -PE (Miltenyi Biotech, 130-111-621) by Cytoflex Flow Cytometer analysis. In the co-culture experiments, the death of CD19+ RAJI cells after 48 h and the CD25 activation (IL2RA, IL-2 receptor alpha chain) and CD107a (marker for degranulation of lymphocytes) cytotoxic de-granulation biomarkers of CAR-T cells and control T cells in CD3+ T cells were analyzed by flow cytometry. Survival analysis of CAR-T cells was performed to control the cell viability of RAJI cells and CAR-T cells. Trypan blue (Biological Industries, #03-102-1B) was applied to identify and count surviving cells. Cell counting and viability analysis were performed with the BIO-RAD TC20 Automated Cell Counter.

The viability of T-2-treated cell samples was evaluated via two different methods. The first method was the trypan blue dye exclusion test. Analysis of viability via this method was performed using the BIO-RAD TC20 automated cell counter (Hercules, CA, USA), according to the manufacturer’s protocol. Cell viability was expressed as a percentage relative to the untreated (control) cells, defined as 100%. The second test was based on measuring cell metabolic activity, which reduces the tetrazolium dye MTT to its insoluble formazan. During this assay, the MTT (0.5 mg/mL) was added to all cell samples and incubated for 4 h at 37 °C. Next, the MTT solution was discarded carefully, and the formed formazan crystals were dissolved in DMSO. The amount of formed formazan crystals was measured calorimetrically at a wavelength of 570 nm with background subtraction at 630 nm on Microplate Reader—BioTek Synergy HT (BioTek Instruments, Winooski, VT, USA). Cell viability was expressed as a percentage relative to the untreated (control) cells, defined as 100%. Half maximal effective concentration (EC₅₀) parameters were calculated using “Quest Graph™ EC500 Calculator” (Bioquest Inc., San Francisco, CA, USA, https://www.aatbio.com/tools/ec50-calculator (accessed on 19 March 2022)).