Pulp cells were seeded in 96-well plates at a density of 1 × 104 cells per well and cultured for 24 h. Subsequently, the cells were exposed to HDPs, MTA Repair HP extract, and a combination of both. The cell cultures were then incubated at 37ºC and 5% CO2 for 24 and 48 h to allow for cellular proliferation. After the specified experimental time points, cell proliferation was assessed using Trypan Blue stain (Sigma). To perform this evaluation, the cells were detached using trypsin (0.025%) and EDTA (0.01%) solution78 (link). Following detachment, a 0.4% solution of Trypan Blue stain (Sigma) was added to the cell suspension and incubated for 1 min. The stained cells were then immediately counted using a Neubauer chamber (Brand GmbH, Wertheim, Germany) under a microscope at 400X magnification. The test was performed in technical triplicate and three individual replicates, conducted on different days, of each sample at each experimental time and compared with the initial cell number of the experiment.
Trypan blue stain
Manufactured by Merck Group
Sourced in United States
Trypan blue stain is a vital dye used in cell viability assays. It is a blue azo dye that selectively colors dead cells or cells with damaged membranes blue, while live cells with intact membranes remain unstained. This dye is commonly used to differentiate between viable and non-viable cells during microscopic examination.
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Lab products found in correlation
Cell countess, by Thermo Fisher Scientific (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Anti cleaved caspase 3 antibody, by Merck Group (2 mentions)
Giemsa, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Minimum essential medium (mem), by Wisent (1 mentions)
Collagen type 1, by Corning (1 mentions)
Universal mycoplasma detection kit, by American Type Culture Collection (1 mentions)
Advanced rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
100 μm cell strainer, by Thermo Fisher Scientific (1 mentions)
Stomacher lab blender, by Seward Medical (1 mentions)
Cd8 pe, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Fc500 flow cytometer, by Beckman Coulter (1 mentions)
Ficoll histopaque 1077 gradient, by Merck Group (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Countess cell counter, by Thermo Fisher Scientific (1 mentions)
30 protocols using trypan blue stain
1
Evaluating Pulp Cell Proliferation
2
Osteocyte Culture and Quantification
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MLO-Y4 osteocytes (courtesy of Dr. Bonewald, Indiana University School of Medicine) are cultured in growth media composed of 2.5% calf serum (CS, Gibco, USA), 2.5% fetal bovine serum (FBS, Gibco, USA), 1% penicillin-streptomycin (PS, Gibco, USA), and 94% Alpha Minimum Essential Medium (MEM) (WISENT, Canada). Cells are seeded during passage 29 at 105 cells per 100 mm diameter collagen-coated (0.15 mg/ml Type I collagen (Corning, USA)) culture dishes and expanded until they achieve 80% confluency. The cells are then transferred onto collagen-coated experimental slides (75x25 mm) at a density of 500k cells per slide for overnight incubation till they reach 80% confluence again before imaging. MLO-Y4 cells are passaged between P29 and P35 while maintained in an incubator at 37 ˚C and 5% CO2. Cell death was quantified using Trypan Blue Stain (Sigma-Aldrich, USA) and counted under a standard light microscope.
3
Mycorrhizal Colonization of Sunflower Roots
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For each applied treatment, four sunflower roots were evaluated for the level of mycorrhizal colonization with R. irregularis at 28 dpi. Small segments (1 cm) of each root were prepared and treated with trypan blue stain, as stated by Phillips and Hayman [25 (link)] (Sigma-Aldrich, Saint Louis, MO, USA). Mycorrhizal colonization was estimated in forty root segments of each treatment while using a light microscope as reported by Trouvelot et al. [26 ].
4
Culturing Human Breast Cancer Cell Lines
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Human breast cancer cell lines MDA-MB-231 and MCF-7 (ATCC; Manassas, VA, USA) were cultured in Advanced RPMI 1640 medium (Gibco; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) in a humidified 5% CO2 incubator at 37 °C. Media were changed every 3 days, once ~80% confluent, cells were harvested with TrypLE Express (Life Technologies; Carlsbad, CA, USA) solution and counted using Trypan blue stain (Sigma Aldrich; St. Louis, MO, USA) and a Cell Countess automated hemocytometer (Life Technologies). Cells were routinely tested for Mycoplasma contamination using the PCR-based Universal Mycoplasma Detection Kit (ATCC). Cell lines were authenticated by short tandem repeat DNA analysis and compared to the ATCC STR profile database (DDC Medical; Manassas, VA, USA).
5
Isolating Lung and Spleen Mononuclear Cells
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Collected tissues (lung and spleen) were homogenized with a stomacher lab blender (Seward, England) into single‐cell suspensions in cold SB. Red blood cells (RBCs) were removed from samples by suspension in appropriate amount of RBC lysis buffer (Sigma‐Aldrich, USA) for 3‐5 minutes and washed with SB. Samples were then filtered with 100‐μm cell strainers (Fisher Scientific, China). To isolate mononuclear cells from the lungs, we used Ficoll gradient Histopaque‐1077 (Sigma‐Aldrich, USA) as previously described.16 Isolated cells from the lungs and spleens were counted with trypan blue stain (Sigma‐Aldrich, USA) using a Neubauer haemato‐cytometer chamber (VWR Scientific, USA). For T cell subset determination, isolated cells were stained with the following antibodies: CD3‐FITC or APC, CD4‐PE/CY7 or APC, and CD8‐PE (Biolegend, USA). Stained samples were analysed with a flow‐cytometer FC500 (Beckman Coulter Inc, USA).
6
Cell Viability Assessment with Trypan Blue
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After treatments cells were harvested, resuspended in cell growth medium, and diluted 1:1 with 0.4% trypan blue stain (Sigma-Aldrich). Stained and unstained cells were counted using a hemocytometer.
7
Quantifying Cancer Cell Survival and Apoptosis
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Single cell suspensions were plated in 6-well plates (500 cells/plate for Pt45P1, 750 cells/plate for PANC-1). After 1 day, cells were treated for 24 hours with gemcitabine. Fresh medium was replaced every 48h. After 10-12 days, cells were fixed in methanol for 10 min, stained overnight with 5% Giemsa (Sigma-Aldrich), washed in PBS and dried. Pictures were taken using a digital camera and colonies were counted. For cell death analyses, cells were seeded at 70% confluence and treated as described for 72 h. Cells were then washed in PBS and either trypsinized and incubated with 0.4% Trypan Blue Stain (Sigma-Aldrich) or processed for caspase 3 immunofluorescence using anti-cleaved caspase-3 antibody (1:500; Sigma-Aldrich) as previously described (31 (link),49 (link)). Positive cells were then counted using the Thoma’s chamber (trypan blue) or fluorescence microscopy (caspase 3). Five random fields were chosen for each treatment and at least 200 cells/field were counted.
8
Cell Viability Assessment with Trypan Blue
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For cell viability and determining concentration, Trypan Blue stain (0.4%; Sigma) was mixed with cells in a ratio 1/1 before being counted by a Countess cell counter (Invitrogen). Dead cells were assayed by counting the number floating (unattached) cells that were trypan blue permeable.
9
Isolation and Transformation of Lymphocytes
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The buffy coat layer was removed from lithium heparinized blood samples and washed with RPMI-1640 media from Sigma-Aldrich in Egypt. The cleaned cells were resuspended in 1 mL of RPMI-1640 media containing 10% fetal calf serum (Sigma-Aldrich, Egypt). The number of viable lymphocytes per mL of RPMI was determined using a Neubauer Hemocytometer and trypan blue stain (Sigma-Aldrich; St. Louis, MO, USA). Each sample was tested three times. 10 Set up for lymphocytes were cultured with phytohemagglutinin (PHA) mitogen at a concentration of 10 µg/mL. The plates were incubated for 72 h at 37 °C in an incubator with a 5 percent CO2 tension. Finally, lymphocyte transformation was carried out at 490 nm utilizing methyl thiazolyl tetrazolium reduction techniques [27 (link)]. The supernatant of cultured lymphocytes was used to collect plasma.
10
Coculture of Human ALL Cells with OP9 Stroma
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