Cell light edu apollo643 in vitro kit

Immortalized human HSC cells (LX-2, ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GibcoTM, Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Genetimes, Shanghai, China) as previously described (Kong et al., 2019a (link)). Cells were used typically between 3rd and 5th passages. Cells were plated at the density of 3 × 10⁵ cells/well in 6-well culture dishes. Primary stellate cells were isolated by collagenase P digestion and Density gradient centrifugation as previously described (Lu et al., 2019 (link)). Small interfering RNA (siRNA) targeting human Cdkn2a (5′-ACACCGCUUCUGCCUUUUCTT-3′) and non-silencing RNA were transfected at final concentration of each 50 nM into LX-2 cells using a Lipofectamine RNAi MAX kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. EdU analysis was performed using Cell-Light EdU Apollo643 in vitro Kit according the manufacturer’s instruction (RiboBio, Guangzhou, China). CCK-8 analysis was performed using Cell Counting Kit-8 according the manufacturer’s instruction (Dojindo, Japan). TGF-β was purchased from PeproTech. Prior to TGF-β treatment, LX-2 cells were serum starved overnight. The next day, TGF-β was diluted in serum-free media to treat the cells.

Cells from different groups were dissociated with 0.25% trypsin-EDTA and harvested by centrifugation. Cell proliferation assays were performed using a Cell-Light EdU Apollo643 In Vitro Kit (RiboBio, Cat# C10310-2) following the manufacturer’s guidelines. Briefly, cells were incubated with 10 μM EdU for 2 hours and subsequently fixed in 4% paraformaldehyde. After Apollo 643 staining, cell nuclei were stained with Hoechst 33342, and cell proliferation was detected by a BD FACSCanto™ System (BD Biosciences, USA).
Cell apoptosis was determined using an Annexin V Apoptosis Detection Kit (KeyGEN, Cat# KGF004). Briefly, cells were incubated with 100 μL of binding buffer containing 2 μL of APC-conjugated Annexin V antibody and 1 μL of propidium iodide staining solution for 15 min at room temperature. After incubation, the cells were immediately analyzed by flow cytometry.

A total of 2.5 × 10⁴ cells were cultured with apatinib (20 μM) for 48 h. EdU detection assay was carried out following the standard protocol of the Cell-Light EdU Apollo643 In Vitro Kit (RiboBio, Guangzhou, China). The A549 and H460 cells were incubated with pre-warmed medium containing EdU (50 μM). The cells were then incubated for 2 h, followed by aspirating culture medium and rinsing cells in PBS. After stopping the reaction, cells were fixed in 4% paraformaldehyde. The paraformaldehyde was then removed by washing. Then, the cells were permeated with 0.5% Triton X-100. The permeation solution was then removed by centrifuging the plate and rinsing cells in PBS. 100 μl Apollo was added into the cells and cultured for 30 min. Finally, the cells were cultured with Hoechst 33342 for 30 min to stain the nuclear. The counting of the positive cells was carried out using the CellProfiler software.

PCa cells were digested and seeded in 96-well plates at a density of 3 × 10³ cells per well. When the cells in the 96-well plate grew to 70–80%, 50 μmol/L EdU (RiboBio Biotechnology, Guangzhou, China) was then added to each well and incubated for 2 h, after which the cells were washed twice using PBS and then fixed with 4% paraformaldehyde for 30 min. The cells were incubated with glycine solution for 5 min at room temperature, after which 1 × Apollo dye solution was added, and cells were incubated in a shaker at room temperature and kept away from light for 30 min. DNA staining was performed after the cells were washed 3 times (10 min each) using 100 μl of osmotic agent. Images were obtained using an OBSERVER D1/AX10 cam heat release capacity (HRC) microscope (Zeiss, Oberkochen, Germany). The Cell-Light EdU Apollo643 In Vitro Kit (RiboBio Biotechnology) was used to perform the EdU flow cytometry test.

A colony formation assay and an EdU staining assay were used to assess cell proliferation. For the colony formation assay, cells were seeded in a 6-cm plate at a density of 500/well and cultured in a complete culture medium for two weeks. After discarding the culture solution, cells were fixed with methanol for 10 min, treated with crystal violet dye for 15 min, washed with running water, and dried. Colonies (>50 cells) were finally photographed and counted. For the EdU staining assay, cells were seeded into 96-well plates at a density of 6,000 cells/well and transfected for 24 h. Cells were incubated with the EdU reagent A for 2 h in an incubator at 37°C. Following the manufacturer's instructions of a Cell-Light™ EdU Apollo643 In Vitro kit (C10310-2, RiboBio, Guangzhou), subsequent experimental steps were performed.

EdU staining was conducted using a Cell-Light EdU Apollo643 In Vitro Kit (Riobio, Guangzhou, China). Briefly, transfected cells were plated for 24 h in a 96-well plate. Then cells were incubated with 50 μM EdU solution for 2 h. After stained with a nuclear dye Hoechst 33342 for 30 min, cell proliferation was estimated using a fluorescence microscope (Thermo Fisher Scientific, Rockville, MD, USA).