Cell proliferation was detected by a Cell Counting Kit (7 Sea Biotech, Shanghai, P.R. China). Cells were grown in 96-well plates (1 × 104 per well) and incubated in 37°C with 5% CO2 until the cell confluency rate reached 70%. After transfection with plasmid for 48 h, the cells were incubated for a further 24, 48, and 72 h. CCK-8 (10 μl) solution was added into each well. The absorbance at 450 nm was measured with SUNRISE Microplate Reader (Tecan, Switzerland).
Cell counting kit
Manufactured by 7Sea Biotech
Sourced in China
The Cell Counting Kit is a laboratory equipment designed to quantify the number of cells in a sample. It provides a simple and reliable method for determining cell concentration, which is crucial for various cell-based experiments and applications.
Automatically generated - may contain errors
7 protocols using cell counting kit
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Cell Proliferation Assay with CCK-8
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Cell Proliferation Assay using CCK-8
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Cell Proliferation and Colony Formation
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Cell Counting Kit (7 sea biotech) was applied for the detection of cell proliferation. Cell growth was performed in a 96‐well plate at 1 × 104/well, while cell incubation was performed in the atmosphere with 5% CO2 at 37°C up to 70% cell confluent. After 48 hr of transfection with plasmids, another 24, 48, and 72 hr of incubation was further performed. And then each well was seeded with 10 ml of CCK8 solution. SUNRISE Microplate Reader (Tecan) was used for the measurement of absorbance at 450 nm.
For EDU assay, EDU labeled solutions (KeyGen Biotech) was used. Nuclei staining was carried out using DAPI.
For colony‐formation assays, single‐cell suspensions were first prepared by the trypsinization of different cell cultures and then seeded into 6‐well plates at 150 cells/well for an incubation of 14 days at 37°C. For the calculation of cell number in each colony, the formed visible colonies were subjected to methanol fixation and then 0.5% crystal violet staining. We counted and recorded the colonies with more than 50 cells for statistical analysis.
For EDU assay, EDU labeled solutions (KeyGen Biotech) was used. Nuclei staining was carried out using DAPI.
For colony‐formation assays, single‐cell suspensions were first prepared by the trypsinization of different cell cultures and then seeded into 6‐well plates at 150 cells/well for an incubation of 14 days at 37°C. For the calculation of cell number in each colony, the formed visible colonies were subjected to methanol fixation and then 0.5% crystal violet staining. We counted and recorded the colonies with more than 50 cells for statistical analysis.
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Cell Proliferation Assay by CCK-8
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Cell proliferation was detected by Cell Counting Kit (7 sea biotech, Shanghai, China). Cells were grown in 96-well plate with 1×104 per well and incubated in 37°C with 5% CO2 until cell confluent rate reached 70%. After transfected with plasmid for 48 h, cells were still incubated for 24, 48 and 72 h. 10 µl CCK8 solution was seed into each well. The absorbance at 450 nm was measured with SUNRISE Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland).
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Histamine H1 Receptor Antagonists Modulate ACE2-HEK293T Cell Viability
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Cell Counting Kit (7Sea Biotech, Shanghai, China) was used to determine cell viability. 5 × 103 of ACE2-HEK293T cells seeded into 96-well plates were incubated for 24 h under standard conditions (37 °C and 5% CO2). Then the medium was replaced with serum-free DMEM or DMEM containing histamine H1 receptor antagonists at concentrations of 25, 50, 100, and 200 μM. The total volume in each well was 100 μl. ACE2-HEK293T cells were incubated in these solutions for 24 h followed by treatment with 10 μl of CCK8 in each well for another 1.5 h at 37 °C. The plates were shaken softly before the detection of the optical density at 450 nm (OD450) using a microplate reader (Bio-Rad, USA). At least three independent experiments were performed. The survival rate of ACE2-HEK293T cells was calculated using the following formula:
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CCK-8 Assay for DPSCs Proliferation
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DPSCs from Sham, IANx and IANx + activin B groups were, respectively, cultured in 96‐well plates (2 × 103 cells/well). CCK‐8 assay was carried out for 1, 3, 5, 7, 9 days according to the cell counting kit (7sea biotech, cell counting kit, China) protocol. After 24 hours, 20 μL CCK‐8 reagent was added into every well and incubates cells for 2 hours. The absorbance was measured at 450 nm wavelength with a microplate reader (Epoch; BioTek).
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Cell Proliferation and Migratory Assays
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Cell Counting kit (7 Sea Biotech, Shanghai, China). Cells were grown in 96-well plate with 2x10 3 per well and incubated in 37˚C with 5% CO 2 until cell confluent rate reached 70%. After transfected with plasmid for 48 h, cells were still incubated for 24, 48 and 72 h. 10 µl CCK8 solution was seed into each well. The absorbance at 450 nm was measured with SUNRISE Microplate Reader (Tecan, Group, Ltd., Mannedorf, Switzerland).
Transwell migration and invasion assay. A total of 1x10 4 cells were transfected with miR-30a-5p mimics or inhibitor for 48 h. The transfected cells were then suspended in a 500 µl serum-free medium and seeded onto a Transwell membrane (Corning Inc., Corning, NY, USA) precoated with Matrigel (BD Bioscience; San Jose, CA, USA). The lower chamber was filled with a 500 µl growth medium containing 10% FBS, which served as a chemoattractant. The cells were cultured at 37˚C for 24 h. The non-invading cells on the top well were gently scraped off. Subsequently, 0.4% crystal violet (Sigma, St. Louis, MO, USA) was used to stain the invaded cells on the lower filter side. Cell invasion was evaluated under the microscope (Olympus Corp., Tokyo, Japan). For migration assay, Transwell membranes were not precoated with Matrigel. Other steps are the same as invasion assay.
Transwell migration and invasion assay. A total of 1x10 4 cells were transfected with miR-30a-5p mimics or inhibitor for 48 h. The transfected cells were then suspended in a 500 µl serum-free medium and seeded onto a Transwell membrane (Corning Inc., Corning, NY, USA) precoated with Matrigel (BD Bioscience; San Jose, CA, USA). The lower chamber was filled with a 500 µl growth medium containing 10% FBS, which served as a chemoattractant. The cells were cultured at 37˚C for 24 h. The non-invading cells on the top well were gently scraped off. Subsequently, 0.4% crystal violet (Sigma, St. Louis, MO, USA) was used to stain the invaded cells on the lower filter side. Cell invasion was evaluated under the microscope (Olympus Corp., Tokyo, Japan). For migration assay, Transwell membranes were not precoated with Matrigel. Other steps are the same as invasion assay.
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