Non-inflamed skin on the upper back of AD patients and healthy participants was tape-stripped 10 times, and 40 μg of Der p 2 rec, and an equimolar mix of Der p 2 pep, saline (0.9% NaCl), and water (all sterile) were applied onto four separate nonwoven fabric spots of adhesive strips for patch tests (Curatest®, Lohmann & Rauscher, GER). The adhesive strips were further secured using a water-repellent plaster. Participants were invited back to the clinics 72 h later, and patch test areas were evaluated for inflammation. One 6-mm biopsy was taken from the skin treated with allergen and one from the skin treated with allergen-derived peptides (two biopsies in total per participant), and biopsies were separately stored in sterile, precooled phosphate-buffered saline for a maximum of 20 min until further processing. Skin biopsies were cut into small pieces using a scalpel, and cells were isolated via enzymatic digestion for 1.5 h using the Gentle MACS Whole Skin Dissociation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, GER). Single cells were resuspended in scRNA-seq resuspension buffer (1x PBS +0.04% BSA (w/vol), sterile), and counted using 0.4% trypan blue solution in saline (Corning, United States). Overall, all samples were processed within 3 h from taking the biopsy until processing for next-generation sequencing.
Trypan blue solution
Manufactured by Corning
Sourced in United States
Trypan blue solution is a laboratory reagent used for cell counting and viability assessment. It is a dye that selectively stains dead cells blue, allowing for the differentiation between live and dead cells in a cell suspension. The solution is commonly used in conjunction with a hemocytometer or other cell counting devices to determine the total cell count and the percentage of viable cells in a sample.
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Lab products found in correlation
Penicillin, by Corning (4 mentions)
Streptomycin, by Corning (4 mentions)
Penicilin streptomycin, by Corning (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Corning (2 mentions)
Antibiotic antimycotic, by Capricorn (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Tc2 automated cell counter, by Bio-Rad (1 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
Countess 2 fl reusable slides, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Fc receptor blocking solution, by BioLegend (1 mentions)
Dbcamp, by Merck Group (1 mentions)
Nutridoma cs, by Roche (1 mentions)
Anti cd11b apc clone icrf44, by BioLegend (1 mentions)
Sytox blue, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
26 protocols using trypan blue solution
1
Skin Allergic Response Profiling
2
Expansion and Characterization of UC-MSCs
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UC-MSCs at passage 4 were seeded in six-well plates with a seeding density of 3000 cells/cm2 in LG-DMEM with 1% GlutaMAX™ (Gibco, Grand Island, NY, USA), 1% antibiotic–antimycotic (Capricorn, Ebsdorfergrund, Germany), and 10% FBS (Capricorn, Ebsdorfergrund, Germany), H-hPL, or Fd-hPL. The morphological changes, growth pattern, and confluency of cultured UC-MSCs were observed and captured using an inverted microscope at a magnification of 40×. Five points per well were captured. The length, width, and size of the cells were analyzed using ImageJ software, version 1.53k. Then, the cells were trypsinized with 0.05% trypsin-EDTA (Gibco, Grand Island, NY, USA) when the cell confluency reached 70%–80%. The cells were stained with 0.4% trypan blue solution (Corning®, Manassas, VA, USA) and the number of viable and non-viable cells was counted using a hemocytometer. The population doubling time (PDT) was calculated using the following formula:
where t denotes time in culture; N2 denotes the cell number at the end of the passage; N1 denotes the cell number seeded at the beginning of the passage.
where t denotes time in culture; N2 denotes the cell number at the end of the passage; N1 denotes the cell number seeded at the beginning of the passage.
3
Cell Growth Quantification Protocol
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Cells (5 × 104) were plated in triplicate in 12-well plates. Cells were prepared with Trypan blue solution (430166, Corning Inc.) at 1:1 ratio and then were counted after 24, 48, and 72 hours using a TC2 Automated Cell Counter (1450102, Bio-Rad).
4
Selective Estrogen Receptor Modulation Assay
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Example 9
MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicilin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 2×104 cells/well, and the following day
5
Trypan Blue Viability Assay
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Trypan blue staining was used to assess the percentage of cells that survived after etoposide treatment combined with PRMT5 inhibition or knockdown. For each time point, cells were seeded on 35 mm dishes and treated as described elsewhere. For cell collection, cells were washed with phosphate buffered saline (PBS) and then trypsinized. Cells were pelleted down using centrifugation (350 × g at 4°C for 5 min), and pelleted cells were resuspended in PBS. Equal volume of cells and trypan blue solution (Corning, USA) were mixed for counting of viable cells and dead cells using Countess II FL reusable slides and the Countess II automated cell counter (Thermo Fisher, USA). The average value of live cells from two aliquot measurements was used as the number of surviving cells.
6
Selective Estrogen Receptor Modulation in Breast Cells
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Example 9
MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicillin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 2×104 cells/well, and the following day Compound 1
7
Cytotoxicity Assay for Reovirus Infection
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L cells (2 x 105 cells/well) or Caco-2 cells (4.2 x 105 cells/well) in complete media were seeded in 12-well plates and incubated until reaching ~ 90% confluency. Cells were adsorbed in triplicate with media alone (mock), with three clones of T1L or T3D at an MOI of 1 PFU/cell (L cells) or 5 PFU/cell (Caco-2 cells). Supernatants were aspirated and replaced with serum-free media post adsorption. Every 24 h for a total of 96 h, cells were gently trypsinized at 37°C and collected via centrifugation at 100 × g. Cells were resuspended in equivalent volumes of PBS without Ca2+ or Mg2+ and 0.4% trypan blue solution (Corning), incubated for 3 min at room temperature, and then manually quantified in duplicate using a hemacytometer and a compound light microscope. Trypan-positive cells are considered to have a disrupted plasma membrane.
8
Evaluating Compound 1 in Breast Cancer Cell Lines
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Example 9
MCF-7 cells were maintained in high glucose DMEM (Corning cellgro, 15-013-CM) supplemented with 10% FBS, 1× L-Glutamine (29.2 mg/mL), and Penicilin Streptomycin (10,000 I.U./mL Penicillin; 10,000 μg/mL Streptomycin; Corning, 30-009-CI). These cells were plated into 24-well plates at 2×104 cells/well, and the following day Compound 1
9
Detailed Flow Cytometry Protocol
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DMSO, ATRA, CA, dbcAMP, PMA, fMLF and NBT were purchased from Sigma; Nutridoma-CS from Roche, Trypan Blue solution from Corning, G-CSF from Applied Biological Materials (abm), FLPEP (catalog # F1314); Sytox Blue (catalog #S11348), TO-PRO3 Ready Flow dead cell stain (catalog #R37170), and NucRed Dead 647 (catalog #R37113) from Life Technologies; and Anti-CD11b-APC (Clone ICRF44, catalog # 301309), isotype control mouse IgG1κ (Clone MOPC-21, catalog # 400107) and Fc Receptor Blocking Solution (catalog # 422302) from Biolegend.
10
Cell Viability Assessment of CAPE Treatment
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FaDu, SNU-1041 and IHOK cells at 50% confluency were treated with vehicle or CAPE. Trypan blue solution in a concentration of 0.4% (Corning, Inc.) was used to stain the cells at a 1:1 ratio at room temperature (RT). Immediately, after staining, cell viability was evaluated automatically using a CytoSMART cell counter (Corning, Inc.).
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