We employed a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Beijing Solarbio Science & Technology Co., Ltd.) to determine cell viability, with the procedures conducted as per the instructions. Briefly, cells were planted into the wells of 96-well plates and allowed to grow until reaching approximately 75% confluence. After the designated treatment, MTT solution with a concentration of 5 mg/mL was introduced into each well for a 4-h cultivation at 37 °C. DMSO (50 μL) was added to each well after solution removal, followed by absorbance (490 nm) measurement utilizing a microplate reader (Thermo Fisher Scientific).
Mtt assay kit
Manufactured by Solarbio
Sourced in China
The MTT assay kit is a colorimetric assay that measures the activity of enzymes that reduce the tetrazolium dye MTT to its insoluble formazan, creating a purple color. The assay can be used to assess cell viability, proliferation, and cytotoxicity.
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Lab products found in correlation
Small interfering rna sirna against nlrp3, by GenePharma (3 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
6 ohda, by Merck Group (2 mentions)
Anti nlrp3 catalog no orb101128 antibody, by Biorbyt (2 mentions)
Rabbit igg catalog no sa00001 2, by Proteintech (2 mentions)
Mouse igg catalog no sa00001 1, by Proteintech (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Trypan blue, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by Maclin Biochemical Technology (1 mentions)
Rabbit anti human bcl 2, by Abcam (1 mentions)
Rabbit anti human bax, by Abcam (1 mentions)
Crystal violet solution, by Solarbio (1 mentions)
Goat anti mouse secondary antibody, by Abmart (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Microplate reader, by Tecan (1 mentions)
Multiwall plate reader, by Agilent Technologies (1 mentions)
26 protocols using mtt assay kit
1
Assessing Cell Viability via MTT Assay
2
Evaluating HRGEC Proliferation and Viability
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The proliferation of HRGECs was assessed by a MTT assay kit (Solarbio, Beijing, China). Cells were seeded at 5000/well on a 96-well plate. At 12 and 24 h after treatment, the cells were washed with PBS and incubated with 10 μl of MTT solution in each well for 4 h. Then 110 μl formazan dissolving solution was added to each well to solubilize the crystals. The colorimetric intensity was measured at 490 nm by a plate reader (Molecular Devices, Sunnyvale, CA, USA). Trypan blue exclusion assay were performed to determine the viability of HRGECs. Cells were washed by PBS and incubated in 0.05% trypsin at 37 °C for 5 min. After dispersing, the single-cell suspension was diluted by 9:1 in 0.4% trypan blue (Solarbio, Beijing, China). Using a hemocytometer, the stained and stain-free cells were counted under an optical microscope.
3
Cell Viability Evaluation using MTT Assay
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Cell viability was evaluated using MTT Assay Kit (Solarbio, Beijing, China). Briefly, MDA-MD-231 and E0771 cells were seeded into 96-well plates with a density of 5 × 103 cells per well. About 48 h after incubation with exosomes or macrophage supernatant, 0.5 mg mL−1 MTT was introduced and further incubated for 4 h. Then, the supernatant of medium was carefully removed and 150 μL dimethyl sulfoxide (DMSO) was added to dissolve the generated formazan. The absorbance at 490 nm was detected by a microplate reader (Bio-Rad). All experiments were performed for three repetitions.
4
Cervical Cancer Cell Line Cultivation
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Human cervical cancer cell lines, SiHa (CSTR:19375.09.3101HUMTCHu113) and HeLa (CSTR:19375.09.3101HUMTCHu187), were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). Both cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM; Gbico, NY, USA) containing 4.5 g/L glucose, 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% penicillin-streptomycin (Sigma-Aldrich, MS, USA). Cells were incubated at 37°C, 5% CO 2 atmosphere and 95% relative humidity. Cells were washed with PBS and substituted with indicated culture medium every 2-3 days. 0.25% trypsin containing EDTA was applied to passage expansion. The major reagents and antibodies used in this study included dimethyl sulfoxide and PTS (Macklin, Shanghai, China) as well as the following antibodies: rabbit anti-human MICA/B, rabbit anti-human p53, rabbit anti-human bcl-2 and rabbit anti-human Bax (all obtained from Abcam, UK), rabbit anti-human PI3K (110α), rabbit anti-human phosphorylated PI3K (p-PI3K) p55 (Tyr199), rabbit anti-human AKT, rabbit anti-human p-AKT (Ser473), goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody (all obtained from Abmart, Shanghai, China). SAHA (molecular weight, 264.32) was purchased from Sigma (No. 149647-78-9). 0.5% crystal violet solution and MTT assay kit were obtained from Solarbio Life Sciences (Beijing, China).
5
Hemolytic and Cytotoxic Effects of UiO-66 Nanoparticles
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Fresh New Zealand rabbit blood was collected, centrifuged at 1500 rpm for 10 min, and washed with PBS five times to separate the red blood cells. The isolated red blood cells were then mixed with UiO-66 NPs at final concentrations of 25, 50, 100, 200, and 400 µg mL−1. Red blood cells treated with 1% Triton X-100 served as positive controls. After incubation at room temperature for 2 h, the cells were centrifuged at 1500 rpm at 4 ℃ for 5 min, and each treated supernatant (100 µL) was added to a new 96-well plate. The absorbance at 492 nm was measured using a microplate reader (Tecan Trading AG, Mannedorf, Switzerland), and the hemolysis rate was analyzed. The MTT assay kit (Solarbio, M1020) was used to detect the toxic effect of UiO-66 NPs, ZrCl4 or TPA in cells at 37 ℃ for 48 h according to the manufacturer’s instructions.
6
Cytotoxicity Evaluation of LPC
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Effects of LPC on viability of NCM460, CT26, HCT116 cells were measured by a colorimetric assay using MTT assay kit (Solarbio, China). NCM460, CT26, HCT116 cells were seeded at a density of 1 × 103/well in a complete growth medium in 96-well plates for 4 duplicates. The cells were incubated with the test compounds for 24 h before the MTT assay. Then, MTT (0.5 mg/mL) was added to each single well with a further incubation for 4 h. Finally, the formazan was dissolved with 100 μL of DMSO and then analyzed in a multiwall plate reader at 570 nm (BioTek Instruments, USA).
7
Quantifying Amino Acid Uptake in Asct2-Manipulated Cells
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HT22 cells were plated into 3 wells of a 6-well plate and infected by lentiviruses which express Asct2 (overexpression), Asct2-specific shRNA (Lenti-1127) (downregulation) or scramble shRNA (control), respectively. After 48 h of infection, cells were incubated in a medium containing 2 μg/ml of puromycin for 72 h when only successfully infected (GFP positive) cells were alive. Further, cells were incubated in a medium containing 1 μg/ml puromycin to expand the culture. To determine the D-serine uptake, cells were plated in a 96-well-plate (5 × 104/well, 3 wells for each infected cell line) to culture for 24 h, and then the medium was changed to 0.1 ml complete medium containing 7 μM D-serine for another 24 h. After that, the medium was collected for HPLC examination, and the rest of the cells were used to measure cell metabolic activity with a MTT assay kit (Solarbio, Beijing, China) following the manufacturer's protocol. The uptake of amino acids was calculated as follows:
The activity of the control was defined as 100%.
The activity of the control was defined as 100%.
8
Neuroprotective Effects of Astragalus in 6-OHDA-Induced PD
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Astragalus was bought from Jianlian Pharmacy (Jinan, China). 6-OHDA was obtained from Sigma-Aldrich, USA (Lot No. MKCC1473). Dulbecco's modified Eagle's medium (DMEM) (Lot No. 8118299, Thermo Fisher Scientific, China) and fetal bovine serum (FBS) (04-001-1ACS, Biological Industries, Israel) were used for daily cell culture. Ethylenediaminetetraacetic acid (EDTA)-free trypsin (Cat. No. T1350) and MTT assay kit were provided by Solarbio (Beijing, China). The assay kits for measuring ROS and mitochondrial membrane potential (MMP), and Hoechst 33342/PI regent were obtained from a biotechnological company of Beyotime (Shanghai, China). Annexin V-FITC/PI Apoptosis Detection Kit (Cat No. A211-02) was bought from Vazyme, China. All the other chemical reagents in this experiment were commercially provided separately.
9
Cytotoxicity Evaluation of Osteoblasts
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In vitro cytotoxicity was measured by MTT assays with MC3T3-E1 osteoblasts, which were purchased from the American Type Culture Collection (Manassas, VA, United States). The cells were grown in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (all Sigma-Aldrich, MO, United States). The cells were incubated at 37°C and 5% CO2. The experiment was conducted with extracts of all groups. Cells were seeded into 96-well cell culture plates at a density of 104 cells/well and routinely incubated. After 24 h, the medium was removed and replaced with the prepared extracts, and culture was continued for another 24 h. Cell viability was tested using the MTT assay kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. To display the cell viability more intuitively, the live/dead staining was carried out with Calcein-AM/PI Double Stain Kit (Solarbio, Beijing, China). Live cells were stained in green by Calcein-AM, whereas dead cells were stained red by PI. The staining results were captured under an inversion fluorescence microscope (BX51WI; Olympus, Tokyo, Japan).
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In vitro cytotoxicity was measured by MTT assays with MC3T3-E1 osteoblasts, which were purchased from the American Type Culture Collection (Manassas, VA, United States). The cells were grown in DMEM with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (all Sigma-Aldrich, MO, United States). The cells were incubated at 37°C and 5% CO2. The experiment was conducted with extracts of all groups. Cells were seeded into 96-well cell culture plates at a density of 104 cells/well and routinely incubated. After 24 h, the medium was removed and replaced with the prepared extracts, and culture was continued for another 24 h. Cell viability was tested using the MTT assay kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. To display the cell viability more intuitively, the live/dead staining was carried out with Calcein-AM/PI Double Stain Kit (Solarbio, Beijing, China). Live cells were stained in green by Calcein-AM, whereas dead cells were stained red by PI. The staining results were captured under an inversion fluorescence microscope (BX51WI; Olympus, Tokyo, Japan).
10
Neuroinflammation Modulation Protocol
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EA (purity > 95%), LPS (Escherichia coli O111:B4), 6-hydroxydopamine (6-OHDA), and apomorphine hydrochloride were obtained from Sigma-Aldrich (St. Louis, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-1β, and IL-18 were bought from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The MTT assay kit was from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Small interfering RNA (siRNA) against NLRP3 was purchased from GenePharma (Shanghai, China). Anti-CR3 complement receptor (OX-42 Catalog No. Ab1211) and tyrosine hydroxylase (TH, Catalog No. Ab113) antibodies were bought from Abcam (Cambridge, MA, USA). Anti-caspase-1 (Catalog No. 22915-1-AP), ionized calcium-binding adapter molecule-1 (Iba-1, Catalog No. 10904-1-AP), β-actin (Catalog No. 20536-1-AP), TNF-α (Catalog No. 17590-1-AP), IL-1β (Catalog No. 66737-1-Ig), IL-18 (Catalog No. 10663-1-AP), rabbit IgG (Catalog No. SA00001-2), and mouse IgG (Catalog No. SA00001-1) antibodies were purchased from the Proteintech Group (Chicago, IL, USA). Anti-NLRP3 (Catalog No. orb101128) antibody was purchased from Biorbyt (Cambridge, United Kingdom).
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