Cellstain Double Staining Kit (Dojindo, Tokyo, Japan) was used to test cell viability as described previously [9 (link)]. Briefly, seeded cells were stimulated according to specific conditions. Afterward, propidium (4 μM) [12 (link)] and calcein-AM (2 μM) were used to stain cells. Ten minutes later, the live and dead cells were discriminated. The living cells hydrolyze calcein-AM by intracellular esterase, generating green fluorescence. In contrast, the dead cells showing red fluorescence. The cell images were recorded with an Olympus CCD camera attached to the immunofluorescent microscope (IX71S1 F-2; Olympus, Tokyo) under the identical settings.
Cellstain double staining kit
Manufactured by Dojindo Laboratories
Sourced in Japan
The Cellstain Double Staining Kit is a laboratory product designed for the simultaneous staining of live and dead cells. It provides a reliable method for distinguishing between viable and non-viable cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ix71s1 f 2, by Olympus (2 mentions)
Ccd camera, by Olympus (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Polyvinyl alcohol (pva), by Fujifilm (1 mentions)
Recombinant human insulin, by Fujifilm (1 mentions)
Dna quantification kit, by Merck Group (1 mentions)
Sodium dihydrogen phosphate nah2po4, by Fujifilm (1 mentions)
L cysteine hydrochloride monohydrate, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
U rfl t, by Olympus (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Eclipse ti u fluorescent microscope, by Nikon (1 mentions)
Biozero bz x700 microscope, by Keyence (1 mentions)
Cell counting kit, by Dojindo Laboratories (1 mentions)
Eosin alcohol solution, by Muto Pure Chemicals (1 mentions)
Hyclone fetal bovine serum fbs, by Thermo Fisher Scientific (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
14 protocols using cellstain double staining kit
1
Cell Viability Evaluation Using Fluorescent Stains
2
PLGA-Based Insulin Delivery System
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PLGA (copolymer composition ratio of 50 : 50, weight average molecular weight of 20 kDa, and inherent viscosity of 0.187 to 0.229 dL/g), methylene chloride (CH2Cl2), polyvinyl alcohol (86–90 mol% hydrolysis), recombinant human insulin, hydrochloric acid (HCl), sodium hydroxide (NaOH), absolute ethanol (99.5%), N-hydroxysuccinimide esters (NHS), 25% glutaraldehyde solution, and sodium dihydrogen phosphate (NaH2PO4) were obtained from Wako Pure Chemicals Ltd., Japan. L-cysteine hydrochloride monohydrate (minimum 98%), ethylene diamine tetra acetic acid (EDTA), papain, DNA quantification kit, Dulbecco's Modified Eagle's Medium (DMEM), growth supplements, and antibiotics were obtained from Sigma-Aldrich, USA. Phosphate buffer saline (10x, pH = 7.4) was obtained from Nacali Tesque Inc., Japan. Porcine collagen type-1 was obtained from Nitta Gelatin, Japan. 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC/EDAC) was obtained from Peptide Institute Inc., Japan. Cellstain Double Staining Kit was obtained from Dojindo Laboratories, Japan. Micro BCA protein assay Kit was obtained from Pierce Biotechnology, USA. All the materials in this study were used as received without further purification. Molecular biology grade milli-Q water from millipore water system (Millipore Corporation, USA) was used for preparation of all the solutions and reagents.
3
Live/Dead Chondrocyte Viability Assay
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Live/dead staining was performed to evaluate cell viability of chondrocytes in the hydrogels by using Cellstain Double Staining Kit (Dojindo Laboratories, Tokyo, Japan). After one and seven days of culture, the cell-laden hydrogel disks were washed with PBS and incubated with serum-free medium containing calcein-AM (2 µM) and propidium iodide (4 µM) for 15 min. After that, the live cells and dead cells in the interior areas of hydrogels were observed under a fluorescence microscope (U-RFL-T; Olympus, Tokyo, Japan).
4
Chondrocyte Viability Evaluation in Hydrogels
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Live/dead staining was performed to evaluate cell viability of chondrocytes in the hydrogels by using Cellstain Double Staining Kit (Dojindo Laboratories, Tokyo, Japan). After UV crosslinking and 14 days of in vitro culture, the cell-laden hydrogel disks were washed with PBS three times and incubated with serum-free medium containing calcein-AM (2 µM) and propidium iodide (4 µM) at 37 °C for 15 min. The stained cells were observed using a confocal microscope (Zeiss LSM 510 Meta).
5
Measuring Brain Vessel Cell Viability
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For cell viability assay, the isolated brain blood vessels were stained using CellStain Double Staining Kit (Dojindo) according to the manufacturer’s protocol. For TUNEL staining, DeadEnd Fluorometric TUNEL System (Promega) was used according to the manufacturer’s protocol.
6
Islet Viability Evaluation via Cellstain Staining
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The Cellstain Double Staining Kit (Dojindo, Kumamoto, Japan), consisting of calcein acetoxymethyl and propidium iodide, was used to analyze islet viability according to the manufacturer's instructions. Samples were observed on an Eclipse Ti–U fluorescent microscope (Nikon, Tokyo, Japan). Islet viability was determined by counting viable (yellow-green) and non-viable (red) cells. The degree of cell viability was assessed as previously described [25 (link)]. Total viability was calculated by dividing the number of viable cells by the total number of cells assessed.
7
Pv11 Cell Viability and Proliferation
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After rehydration in fresh complete IPL-41 medium, Pv11 cells were collected at 1, 3, 5, and 7 d following rehydration and stained with a Cellstain Double Staining Kit (DOJINDO), following the manufacturer’s instructions. Bright-field images and calcein-AM fluorescence were visualized under a Biozero BZ-X700 microscope (Keyence), and Pv11 cells were quantified using BZ-H3C call count software (Keyence). One day after rehydration, cell viability was estimated from the number of calcein-AM–positive (live) cells, divided by the total number of cells observed by bright-field microscopy. Cell proliferation was assessed from the total number of cells counted every 2 d by bright-field microscopy. Each measurement was made with 12 replicates.
8
HPBCD-Assisted Cell Culture Examination
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HPBCD was kindly donated by Nihon Shokuhin Kako Co., Ltd. (Tokyo, Japan). Mayer's hematoxylin, 1% eosin alcohol solution, and mounting medium for histological examination (malinol) were from MUTO Pure Chemicals (Tokyo, Japan). Dulbecco's modified Eagle's medium and F-12 medium were obtained from Gibco-Life Technologies (Life Technologies Japan, Tokyo, Japan). HyClone™ fetal bovine serum (FBS) was purchased from Thermo Scientific (Logan, UT, USA). The cell counting kit and Cellstain® Double Staining Kit were obtained from Dojindo Laboratories (Kumamoto, Japan). All other reagents and solvents were of reagent grade. De-ionized and distilled bio-pure grade water was used throughout the study.
9
Assessment of Cell Viability
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Calcein‐AM and propidium iodide (PI) were used to identify live and dead cells, respectively (Cellstain Double Staining Kit; Dojindo Laboratories). An assay solution consisting of calcein‐AM and PI was prepared by adding 10 μL of 1 mmol/mL calcein‐AM stock solution and 15 μL of 1.5 mmol/mL PI solution to 5 mL of HBSS(+). NHEK‐Ad cells were pre‐cultured for 24 hours and washed twice with HBSS(+) before treatment with 1 mL/well of 0.5 mM C8K, C18:1K, SLES or SLS for 5 minutes at room temperature. The cells were washed three times with HBSS(+) and stained with 0.5 mL of the assay solution for 15 minutes.18 The cells were washed with HBSS(+) three times and fresh HBSS(+) was added to each well. The double‐stained cells were assessed by fluorescence microscopy (Biorevo BZ‐9000, Keyence Corporation) (Figure 3 ).
10
Quantifying Live Cells Using Fluorescent Dyes
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Cells were incubated with Calcein-AM dye by using the Cellstain-Double Staining Kit (Dojindo, #C542) and Hoechst (Life, #H3570) for 15 min at 37 °C. Living cells stained green were observed using the Operetta CLS High-Content Analysis System (Perkin Elmer, USA).
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