Trypan blue exclusion and lactate dehydrogenase (LDH) cytotoxicity assays were used to assess TCS cytotoxicity on Jurkat cells. In the trypan blue exclusion assay, 1 million cells were plated into each of 3 wells of a 24-well, flat-bottom cell culture plate (Costar) for TCS (Sigma) treatment and 3 wells for the control. Immediately after plating, BT and 2X TCS doses were added to respective wells. Next, cells were incubated for 30 minutes at 37°C/5% CO2. After the incubation, a small sample was taken from each well and mixed 1:1 with trypan blue dye (0.4%, Lonza) evenly. Cells were counted using a hemocytometer for viability and TCS-treated cells were normalized to the control. LDH cytotoxicity methods were those of Hutchinson et al., 2011 (link) using a cytotoxicity detection kit (Roche). However, the lysis solution was added to the “high control” in the final 15 minutes of the 1-hour TCS exposure instead of an additional 15 minutes after the 1-hour TCS exposure (Palmer et al., 2012 (link)).
Trypan blue dye
Manufactured by Lonza
Sourced in United Kingdom
Trypan blue dye is a commonly used vital stain for the identification of dead cells in a cell suspension. It is a blue azo dye that can permeate the cell membrane of dead, damaged, or dying cells, staining them blue. This allows for the visual distinction between viable (unstained) and non-viable (blue-stained) cells under a microscope.
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7 protocols using trypan blue dye
1
Trypan Blue and LDH Cytotoxicity Assays
2
C2 Mouse Skeletal Myoblast Culture
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C2 mouse skeletal myoblasts (passages 8–12) (Yaffe and Saxel 1977 (link)) were grown in T75 flasks in a humidified 5% CO2 atmosphere at 37 °C in growth medium (GM), composed of: DMEM with Glutamax supplemented with 10% hiFBS, 10% hiNCS, penstrep and additional l -glutamine, at final concentrations of 10000 U ml−1 and 2 mM respectively, until 80% confluency was attained. Experiments and differentiation were initiated following washing with phosphate buffered saline (PBS), by transferring to low serum-containing differentiation medium (DM; DMEM plus glutamax, supplemented with 2% HS, penstrep and additional l -glutamine (supplemented as above)) in the absence or presence of specific cytokines, growth factors, lipids and signal inhibitors described below. C2 myoblasts, upon serum withdrawal, are able to undergo spontaneous differentiation into myotubes and do not require growth factor addition to stimulate the process (Florini et al. 1991 (link)). Adherent cells following trypsinisation, and detached cells in the supernatant, were counted using a haemocytometer in the presence of trypan blue dye (Bio Whittaker, Wokingham, England) to assess viability and cell number.
3
Cytokine and Sunitinib Effects on Breast Cancer Cell Viability
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Breast cancer cells were seeded at a density of 5 × 104/mL in 12-well cluster plates and incubated overnight. The next day, the cells were treated with cytokines (5 ng/mL each TNF-α and IFN-γ), sunitinib (10 μM), both, or left untreated. After 72 hours of further incubation, cells were harvested by scraping, and 0.002% Trypan Blue dye (Lonza, Walkersville, MD) was added in 30 μL volume of cell suspension. Dye uptake was assessed via flow cytometry using a FlowSight cytometer (Amnis/Millipore) employing a 642 nm laser, and analysis performed using IDEAS version 6.2 software as described previously [12 (link), 13 (link)].
4
Cell Membrane Integrity Assay
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Cell membrane integrity after PcGal16 incubation in the dark, irradiation, or both was determined by the trypan blue dye (Biowhittaker, Walkersville, MD, USA) exclusion test 24, 48 and 72 h after each treatment. Cells with intact membrane were counted on a Neubauer chamber after trypsinization and the cell viability of treated cells was normalized to that of the untreated cells.
5
Cell Sprouting Assay for Angiogenesis
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A cell sprouting assay was performed using HCE cells in Cultrex (Trevigen, Gaithersburg, MD, USA) (reduced and diluted in MEM at 1:1). HCE cells were embedded in Cultrex at 5 × 105 cells/10 μL of spot, then dried in an incubator for 45 min, followed by the addition of media with treatment or control. The cell-spotted plates were then exposed to reduced serum media (0.5% FBS) (Un negative control), Hst5 (50 μM), or 10% FBS (positive control). The Cultrex-embedded spots were then imaged at 40 and 72 h using an inverted light microscope (DMi1, Leica Microsystems, Buffalo Grove, IL, USA) at ×4 magnification. The cellular spot-covered area at each time point was measured using ImageJ v1.47 software (NIH, Bethesda, MD, USA). At 72 h, cells were fixed using methanol for 20 min and stained with trypan blue dye (Lonza, Basel, Switzerland).
6
Optimizing Hybridoma Cell Culture Conditions
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Suspension mouse-mouse hybridoma cell line, SP2/0-Ag14 (ATCC, Manassas, VA) were thawed from liquid nitrogen tank and after a few passages were transferred to modified T-75 flasks to study the effectiveness of the method developed for removing leachables and extractables. 21 ml of complete medium (90% v/v of DMEM supplemented with 10% v/v of FBS) were added to each flask. The seeding density in each T-flask was 6.3 × 104 viable cells/mL. Cell density and viability were determined using hemocytometer and trypan blue dye (Lonza, Walkersville, MD) technique.
7
C2C12 Myoblast Differentiation and Analysis
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Murine skeletal muscle cells were purchased from ATTC (Rockville, MD, USA) and maintained in a humidified 5% CO 2 environment at 37°C. The cells were grown in growth media (GM) supplemented with DMEM media (Sigma, Gillingham, Dorset, UK), 10% Fetal bovine serum (hi FBS; Thermo Fisher, UK), 10% new born calf serum (hi NCS; Invitrogen, GIBCO, Paisley, UK), 1% L-glutamine and 1% of 10,000 U Penicillin/Streptomycin until 80% confluence was attained. Following trypsinisation, C2C12 myoblasts were detached and counted using a haemocytometer in the presence of trypan blue dye (Bio Whittaker, Wokingham, England). Six-well plates were pre-coated with 0.2% gelatine for 5 min at room temperature. Following removal of excess gelatine, C2C12 myoblasts were seeded at 1x10 5 cells/ml in GM. On attaining approximately 80% confluency, cells were washed twice with PBS before culturing with CMs. After 4 days of CMs treatment, C2C12 myoblasts were collected to perform the following the studies: Biochemical and morphological studies; cell cycle analysis, gene expression assay qRT-PCR after 4 days, and Luminex xMAP phosphoproteins magnetic bead arrays after 25 min post transfer to CMs.
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