Colony formation experiments were carried out by plating ~100 cells per well into 6-well dishes. Following a 10-day incubation at 37°C, the number of colonies (> 50 cells) was scored using standard methods [23 (link)]. Cell proliferation was gauged by the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2ʹ-Deoxyuridine (EdU) assays using the MTT Cell Proliferation Assay Kit (Biovision, Wehrheim, Germany) and Cell-Light EdU Apollo488 In Vitro Kit (Ribobio), respectively, as per the accompanying protocols and standard methods [24 (link),25 (link)]. Cell apoptosis was determined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit (BD Biosciences, Cowley, UK) as described elsewhere [12 (link)].
Protocol detail
Cellular Assays for Proliferation, Colony Formation, and Apoptosis
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MTT Cell Proliferation Assay Kit
Cell-Light EdU Apollo488 In Vitro Kit
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit
5 ethynyl 2 deoxyuridine
Annexin v
Apoptosis
Assay
Bromide
Cell
Cell proliferation
Dishes
Fluorescein
Isothiocyanate
Light
Propidium iodide
Corresponding Organization :
Other organizations : Henan Provincial People's Hospital, Henan Forestry Vocational College
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Variable analysis
independent variables
- Cell plating density (approximately 100 cells per well)
- Number of colonies (> 50 cells)
- Cell proliferation (as measured by MTT and EdU assays)
- Cell apoptosis (as measured by Annexin V-FITC/PI assay)
- Incubation time (10 days)
- Incubation temperature (37°C)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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