4000/well cells were seeded on 96-well plates and transfected with siRNAs. Cell proliferation was done after 0, 24, 48, and 72 h by cell counting kit (TransGen Biotech, Beijing, China) at 450 nm.
Cell counting kit
Manufactured by Transgene
Sourced in China
The cell counting kit is a laboratory equipment used to determine the number of cells in a sample. It provides a quantitative measurement of cell concentration.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan skyhigh photometer, by Thermo Fisher Scientific (3 mentions)
96 well culture plate, by SPL Life Sciences (2 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
One step gdna removal and cdna synthesis supermix, by Transgene (1 mentions)
Alexa fluor 488, by Cell Signaling Technology (1 mentions)
Microplate spectrophotometer, by Bio-Rad (1 mentions)
11 protocols using cell counting kit
1
Cell Proliferation Assay with siRNA
2
Proliferative Ability of Senescent Fibroblasts
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To confirm changes in the proliferative ability of senescent fibroblasts treated with PDLLA or HA, 2500 fibroblasts were seeded in each well of a 96-well culture plate (SPL Life Sciences, Pocheon, Republic of Korea), and the in vitro model was manufactured as described in Section 2.2.4 . Cell counting kit (TransGen Biotech Co., Ltd., Beijing, China) was mixed with serum-free DMEM (1:9, v/v; Welgene, Gyeongsan, Republic of Korea), and the mixture was incubated for 4 h at 37 °C in an atmosphere containing 5% CO2. Optical density was measured at 450 nm using a plate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific, Rockford, IL, USA).
3
ATBF1 Regulation in TGF-β Signaling
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The following reagents were purchased from their respective vendors: TGF-β (PeproTech, Inc., Jiangsu, China); anti-ATBF1 primary antibody (Santa Cruz Biotechnology, Shanghai, China); Alexa Fluor® 488 (Cell Signaling Technology (CST), Shanghai, China); 4′,6-diamidino-2-phenylindole (DAPI) (CST); Lipofectamine RNAiMAX (ThermoFisher Scientific, Shanghai, China); Cell Counting Kit (TransGen BiotechCo., Ltd., Beijing, China); One-Step gDNA Removal and cDNA Synthesis Supermix (TransGen Biotech); and TransStart® Top Green qPCR SuperMix (TransGen Biotech).
Primers of ATBF1, E-cad, N-cad, and c-myc were synthesized from Shanghai Generay Biotech Co., Ltd. The sequences of gene primers are listed inTable 1 . Small interfering RNAs (siRNAs) were synthesized from Shanghai GenePharma Co., Ltd. The sequence of siATBF1 is 5′-AGAAUAUCCUGCUAGUACA-3′.
Primers of ATBF1, E-cad, N-cad, and c-myc were synthesized from Shanghai Generay Biotech Co., Ltd. The sequences of gene primers are listed in
4
Cell Viability Assay with CCK-8
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Cell viability was determined using an Cell Counting Kit (CCK-8, Transgen, Beijing, China) according to the manufacturer’s protocol. Briefly, 1.5×104 cells were seeded into each well of a standard 96-well plate and incubated at 37°C with 5% CO2. After overnight culture, the media was removed, and the cells were infected with different virus strains. Infected and uninfected cells were assayed at 12, 24 and 48 h post-infection using CCK8 kit. The absorbance was determined at 490 nm using a microplate spectrophotometer (Bio-Rad, Hercules, CA, USA).
5
Quantifying A431 Cell Proliferation
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A431 cells (4000 per well) cultivated on 96-well plates were transfected with siRNAs, and cell proliferation was detected after 0, 24, 48, and 72 h using a cell counting kit (TransGen Biotech) at 450 nm as described in the manual.
6
Cell Proliferation Assay for Engineered Constructs
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ECs were seeded in 96-well plates (LPS solution, Daejeon, Republic of Korea) at a density of 1 × 103 cells per well and allowed to adhere for 24 h. The cell proliferation of the ECs in response to the PDLLA, PN, or CaHA treatment was confirmed using a cell counting kit (Transgen Biotech Co., Ltd., Beijing, China) once the in vitro model was manufactured, as mentioned in Section 4.2.3 . cell counting kit solution was mixed with serum-free medium (1:9, v/v), the mixture was added to each well, and the cells were incubated for 4 h at 37 °C. The optical density was measured at 450 nm using a microplate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific, Rockford, IL, USA).
7
Huh7 Cell Proliferation Assay
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Huh7 cells were cultured in 24-well plates and transfected with plasmids pcDNA-LRRFIP1 or mock vector. 48 hours after transfection, cells were allowed to grow in 96-well plates (3000 cells/well) and cultured for 24 hours. Cell proliferation was detected using Cell Counting Kit (Transgen, China) at 12 or 24 hours intervals according to the manufacturer’s instruction.
8
Cell Proliferation and Colony Formation Assay
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A431 cells (4000 per well) cultivated on 96-well plates were transfected with siRNAs and cell proliferation was detected after 0, 24, 48, and 72 h using a cell counting kit (TransGen Biotech) at 450 nm as described in the manual. For the colony forming assay, transfected cells were incubated in 6-well plates at 1000 cells per well, which were maintained in DMEM. Medium was replaced 2 times. At day 7, cells were collected after being washed twice with PBS and fixed in 4% paraformaldehyde for 30 min. Finally, the cells were stained with 0.1% crystal violet. Visible colonies were photographed and counted.
9
Assessing Apoptosis in Senescent Macrophages
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To determine the apoptotic capacity of senescent macrophages treated with PDLLA or HA, 5000 murine macrophages (RAW 264.7 cells) were seeded in each well of a 96-well culture plate (SPL Life Sciences, Pocheon, Republic of Korea). Senescence induction of murine macrophages was made as mentioned in Section 2.2.1 ., and senescence-induced murine macrophages were treated with PDLLA and HA by concentration (100, 200, or 400 µg/mL) for 24 h. The cell counting kit (TransGen Biotech Co., Ltd., Beijing, China) mixed with serum-free DMEM (1:9, v/v; HyClone-Cytiva™, Marlborough, MA, USA) was added, and the mixture was incubated for 4 h at 37 °C in an atmosphere containing 5% CO2. Optical density was measured at 450 nm using a plate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific, Rockford, IL, USA).
10
Palmatine Cytotoxicity on RAW264.7 Cells
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Murine RAW264.7 cells were seeded into 96-well plates at a density of 1×104 cells per well and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 for 24 h. Subsequently, palmatine at 0 (control), 128, 256 and 512 μg/mL were added to the culture medium and incubation was continued for 12 h. The activity of cells was measured by Cell Counting Kit (CCK) assay56 (link) according to the manufacturer’s protocol (TransGen Biotech, Beijing, China).
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