Bovine serum albumin (heat shock fraction, pH 7, ≥98%) and rhodamine 6G (Rho) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The glutaraldehyde solution (25% for synthesis) used for crosslinking was purchased from AppliChem GmbH (Darmstadt, German) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride was from Tokyo Chemical Industry (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were from Gibco (New York, NY, USA). The Hoechst 33,342 solution and apoptosis kit were from Invitrogen (Waltham, MA, USA). The Cell Counting Kit-8 was from Enzo Life Sciences (New York, NY, USA). Ultrapure water (Merck, Darmstadt, Germany) was used for all experiments. All other reagents used were of analytical grade.
Cell counting kit 8 (cck8)
Manufactured by Enzo Life Sciences
Sourced in United States
The Cell Counting Kit-8 is a colorimetric assay used to determine the number of viable cells in cell proliferation and cytotoxicity assays. It utilizes a highly water-soluble tetrazolium salt to measure the metabolic activity of cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Opti mem buffer, by Thermo Fisher Scientific (2 mentions)
Dpbs buffer, by Thermo Fisher Scientific (2 mentions)
Opti mem buffer, by Promega (2 mentions)
Cytotox 96 non radio cytotoxicity assay, by Promega (2 mentions)
Multiplate reader, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
Hoechst 33342 solution, by Thermo Fisher Scientific (1 mentions)
Rhodamine 6g rho, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide hydrochloride, by Tokyo Chemical Industry (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Elisa microplate reader, by Tecan (1 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (1 mentions)
Ldh cytotoxicity colorimetric assay kit 2, by Abcam (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Microquant, by Agilent Technologies (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
28 protocols using cell counting kit 8 (cck8)
1
Bovine Serum Albumin-Rhodamine 6G Conjugation
2
Measuring Astrocyte Viability with CCK-8
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The Cell Counting Kit-8 (CCK-8, #ALX-850-039-KI01, ENZO) was used to measure cell viability and proliferation. The culture medium of astrocytes (2 × 105cells/well) cultured in 0.1 mg/mL PDL-cover slip, Ti and GO-Ti in a 24-well plate (SPL) was removed. The cells were then washed twice with 1 × PBS. In each well, 450 μL of astrocyte culture medium and 50 μL of CCK-8 solution was added and reacted at 37°C and 5% CO2 for 3 h. The absorbance was measured at 450 nm using a microplate reader (BIOTEK). The values calculated for cell viability and proliferation were as follows: O.D value of the sample–O.D value of the blank (culture medium + CCK-8 solution).
3
Cell Viability Assay with CCK-8
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Cell viability was determined using the Cell Counting Kit 8 assay (Enzo Life Sciences, Euroclone, Milan, Italy) according to the manufacturer’s instructions. This test is based on the mitochondrial enzyme-dependent reaction of a water-soluble tetrazolium salt for quantifying the number of live cells by producing an orange formazan dye upon bio-reduction in the presence of an electron carrier. Briefly, cell cultures were grown under the different experimental conditions for 24 h before replacing the medium with 100 µL of a 10% (v/v) mixed solution of the WST-8/CCK8 probe in fresh DMEM. Cell viability after 4 h incubation was determined with a spectrophotometric measure of absorbance at 450 nm using an ELISA microplate reader (Tecan, Sunrise, Männedorf Switzerland). The data were normalized to those of the 6-OHDA to measure relative cell viability with respect to CIT and SLNs resuspended after freeze-drying.
4
Cytotoxicity Assessment of cp-asiRNAs
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Example 5
To test the cytotoxicity of cp-asiRNAs, MNT-1, a human melanoma cell line, and HaCaT, a human keratinocyte cell line were treated with cp-asiTYR #4-1 and hydroquinone.
The cp-asiRNA was incubated at 95° C. for 2 minutes and at 37° C. for 1 hour in OPTI-MEM buffer (Gibco). Proper strand annealing of the potential cp-asiRNAs was confirmed by gel electrophoresis.
One day before treatment with cp-asiRNA(4)-1, 5.0×103 MNT-1 cells or 1.0×104 HaCaT cells were seeded into 96 well plates. Immediately before treatment, the cells were washed with 1×DPBS buffer (Gibco), and then cultured in the presence of 1 μM or 3 μM of cp-asiRNATYR(4)-1 in OPTI-MEM buffer for 24 hours, at which point the cytotoxicity level was measured using a CytoTox96 Non-Radio Cytotoxicity assay (Promega) according to manufacturer's instructions. The media was then replaced with the serum-containing media and cell viability was measured using a cell counting kit-8 (Enzo) according to manufacturer's instructions.
As shown in
5
Cell Viability Assay Using WST-8
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A cytotoxicity test was conducted using Cell Counting Kit-8 (Enzo Life Sciences Inc., NY, USA) according to the manufacturer’s instructions. The culture media was replaced with WST-8 cell proliferation reagent [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] for 1.5 h at 37 °C. With its higher sensitivity as a chromogenic indicator for cell viability compared with conventional tetrazolium salts, WST-8 produces a water-soluble formazan dye upon reduction in the presence of an electron carrier. The absorbance of the formazan dye was measured at 450 nm using a microplate spectrophotometer (EMax, Molecular Devices, USA).
6
Comparative ROCK Inhibitor Toxicity
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The relative toxicity of five log concentrations of Y-27632 and fasudil (0.1 μM, 1 μM, 10 μM, 100 μM, and 1000 μM) were tested. U87-MG, JX12, and SMC448 cells were seeded in 96 well plates at a seeding density of 1x104 cells/well (n = 10 for each ROCK inhibitor). The resulting cell viability was measured at 450 nm absorbance using a water-soluble tetrazolium salt-based proliferation assay according to manufacturer’s protocol (Cell Counting Kit-8, Enzo Life Sciences, Farmingdale, NY). The data were normalized to those of the control (group not treated with either inhibitor) to measure relative cell viability.
7
Evaluating the Effects of CLHs on FL83B Cells
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First, the culture method of FL83B cells was slightly modified from the American Type Culture Collection (ATCC) [18 (link)]. The short- and long-term impacts of CLHs on the survivability of FL83B cells in fetal bovine serum (FBS) containing culture system were determined (Fig. 1A ), which could monitor cellular morphology and growth condition compared to those of ATCC. Survival assay (Cell Counting Kit-8; Enzo Life Science, Inc., Farmingdale, NY, USA) and lactate dehydrogenase (LDH) leakage assay (LDH-Cytotoxicity Colorimetric Assay Kit II; BioVision Inc., Milpitas, CA, USA) of cells were conducted according to the commercial manuals, respectively. FL83B cells were seeded in a 96-well plate (20,000 cells/well), incubated for 12 h to attach. The cell survival assay was then taken after another 6- or 12-h incubation under 100 μL 10% FBS-contained media (0 to 4000 mg CLHs/L). Next, 4 h before the harvest time point, the 10 μL CCK-8 was added. Final optical density values were obtained via Synergy H1 Hybrid Reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).
8
Cytotoxicity and Proliferation Assays
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A cytotoxicity test was conducted using the Cell Counting Kit-8 (Enzo Life Sciences Inc., NY, USA). The culture medium was replaced with water-soluble tetrazolium salt (WST)-8 reagent [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]. Then, the cell plate with WST-8 reagent was kept in an incubator containing 5% CO2 for 1.5 h at 37 °C. The absorbance of a water-soluble formazan dye produced by the reagent was measured at 450 nm using an ELISA microplate reader.
The dyeing solution for analysis of proliferation was prepared by mixing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO, USA) solution with a 10% ratio to α-MEM. The culture medium was replaced with the MTT solution. After incubation for 4 h, the solution was removed. To dissolve the dark-blue crystals of MTT formazan, dimethyl sulfoxide (DMSO; Duksan Pure Chemical Co., Ltd., Ansan-si, Korea) was added to each specimen. Absorbance after solubilization of the formazan was measured at a wavelength of 540 nm using an ELISA microplate reader.
The dyeing solution for analysis of proliferation was prepared by mixing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO, USA) solution with a 10% ratio to α-MEM. The culture medium was replaced with the MTT solution. After incubation for 4 h, the solution was removed. To dissolve the dark-blue crystals of MTT formazan, dimethyl sulfoxide (DMSO; Duksan Pure Chemical Co., Ltd., Ansan-si, Korea) was added to each specimen. Absorbance after solubilization of the formazan was measured at a wavelength of 540 nm using an ELISA microplate reader.
9
TMBIM6 Expression Modulates Cell Viability
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To analyze the Sp1 mediated TMBIM6 expression effect on the cell viability during stress, we have generated the constructs by subcloning ORF of TMBIM6-HA from pCDNA3-TMBIM6-HA plasmid into P9 promoter construct and named as P9-TMBIM6-HA, in which P9 promoter drives the TMBIM6-HA expression. Another constructs was generated by subcloning ORF of TMBIM6-HA into Sp1 sites mutated P9 promoter construct and named as mP9-TMBIM6-HA. Cell lines representing different cancer were seeded into 96-well plates at the density of 1 × 104 cells/well. After 12 h of seeding cells were transfected with P9-TMBIM6-HA or mP9-TMBIM6-HA plasmid. After 24 h of transfection cells were treated with different anticancer drugs paclitaxel and cyclophshomide at different concentrations in triplicate for 24 h. After 24 h of treatment, cell viability was assessed using the cell counting kit-8 (Enzo life science, Farmingdale, NY, USA, #ALX-850-039-KI01) by following the manufacturer’s protocol. The data represented is the percentage of viable cells in a well and were normalized to the untreated control wells for each agent.
10
Evaluating PPAR-iMSC-EV Impacts on Hepatocyte Viability
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Primary human hepatocytes (2 × 103 cells/mL) were seeded in 96-well plates and cultured for 4 h in serum-free DMEM at 37 ℃ and 5% CO2. The absorbance (OD value) was measured at 450 nm using a multiplate reader (Thermo Fisher Scientific). The effects of pan PPAR-iMSC-EVs on the viability of primary hepatocytes were evaluated using the Cell Counting Kit-8 (Enzo life sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions.
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