HeLa and Siha cells, at a density of 2x105 cells/well, were seeded and cultured in the 6-well plates overnight. Transfections were performed the following day. Target cells were cultured over the desired time periods. The DNA synthesis rates were measured using an EdU Apollo488 In Vitro Flow Cytometry kit, following the manufacturer’s instructions (Guangzhou RiboBio, Co., Ltd.).
Edu apollo488 in vitro flow cytometry kit
Manufactured by RiboBio
Sourced in China
The EdU Apollo488 In Vitro Flow Cytometry kit is a reagent used for the detection and quantification of cell proliferation in vitro through flow cytometry. The kit contains EdU (5-ethynyl-2'-deoxyuridine), a thymidine analog, which is incorporated into the DNA of proliferating cells. The incorporated EdU is then detected using a fluorescent azide dye, in this case, Apollo488.
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4 protocols using edu apollo488 in vitro flow cytometry kit
1
Cell Proliferation Assay via EdU
2
EdU/PI Staining for Cell Cycle Analysis
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EdU/PI staining was performed as described previously.29 (link) Briefly, VECs (107 cells) were seeded in a 6-well plate and incubated overnight. Then, BFM was added (2.5 mL/well) and the cells were divided into three treatment groups: 100 ng/µL CGS 21680, 100 ng/µL ZM241385, and 1.25 µL DMSO. The treatments were applied for 48 h. The cell-light EdU Apollo 488 in vitro flow cytometry kit (C10338-3, Ribobio) and propidium iodide (PI) were used to detect the cells. The kits were used in accordance with the manufacturer’s instructions.
3
Podocyte Proliferation Assay with TNF-α
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With 96‐well plates, podocytes were seeded at 104 cells/well in 100 μL complete medium. After culture overnight at 37°C with 5% CO2, the cells were exposed to control or TNF‐α at 5 ng/mL for 24 hours, 48 hours, or 72 hours. CCK‐8 (10 μL; Beyotime, Shanghai, China) solution was added into each well and then incubated for another 1 hour. Absorbance was examined at 450 nm by a microplate reader (Bio‐Rad, Hercules, CA).
With BD FACSCalibur flow cytometer (BD Biosciences, CA, USA), cell proliferation was determined by 5‐ethynyl‐20‐deoxyuridine (EdU) incorporation. According to the protocol of EdU Apollo 488 In Vitro Flow Cytometry Kit (RiboBio, Guangzhou, China), EdU of 20 μL was added into each well and the cells received another 2 hours incubation. After collection and centrifugation, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with anti‐EdU working solution for 30 minutes. Finally, EdU‐positive cells were quantified using flow cytometry,
With BD FACSCalibur flow cytometer (BD Biosciences, CA, USA), cell proliferation was determined by 5‐ethynyl‐20‐deoxyuridine (EdU) incorporation. According to the protocol of EdU Apollo 488 In Vitro Flow Cytometry Kit (RiboBio, Guangzhou, China), EdU of 20 μL was added into each well and the cells received another 2 hours incubation. After collection and centrifugation, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with anti‐EdU working solution for 30 minutes. Finally, EdU‐positive cells were quantified using flow cytometry,
4
EdU Incorporation Assay by Flow Cytometry
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1 × 106 MHCC-97H or SMMC-7721 cells were seeded into 6-well plates. 50 μM of Edu from Edu Apollo® 488 In Vitro flow cytometry Kit (RiBoBio) was added into the plates 2 h before harvesting the cells. Cells were collected and centrifuged at 1000 rpm/5 min, and supernatant was removed. For fixation, 4% paraformaldehyde was added into the cells and incubated for 15 min, and washed once by 1 × PBS. Then cells were resuspended in Tris buffer saline with 0.5% Triton X-100 and incubated for 10 min, and washed again with 1 × PBS. Amounts of 500 μl staining solution with Apollo® 488 fluorescent azide was added into cells, incubated for 10 min, and then rinsed twice with Tris buffer saline with 0.5% Triton X-100. All of the cells were analyzed by flow cytometry (BD).
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