We then determined the role of USP22 in cell proliferation using the EdU (5-ethynyl-2′-deoxyuridine) incorporation assay as described previously [6 (link)]. Briefly, USP22-silenced and control GC cells were seeded in 6-well plates and grown for24 h. Then, the cells were incubated with 50 μM EdU (RiboBio, Guangzhou, China) for 2 h and stained with Apollo®567 according to the manufacturer’s instructions. After washing thrice with cold phosphate-buffed saline (PBS), cells were incubated with the DNA staining dye Hoechst 33342 (Sigma) for 10 min. The stained cells were photographed using an inverted fluorescent microscope (Olympus) and six random fields were selected to estimate the total number of EdU-positive cells in each of the samples.
Apollo 567
Manufactured by RiboBio
Sourced in China
The Apollo 567 is a high-performance laboratory equipment designed for various scientific applications. It features advanced technology and superior functionality to facilitate efficient and precise measurements and analysis. The core function of the Apollo 567 is to provide reliable and consistent performance in a wide range of laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
5 ethynyl 2 deoxyuridine (edu), by RiboBio (6 mentions)
Edu c10341 1, by RiboBio (4 mentions)
Hoechst 33342, by RiboBio (2 mentions)
C10341 1, by RiboBio (2 mentions)
Inverted fluorescent microscope, by Olympus (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Olympus (1 mentions)
12 well transwell plate, by Corning (1 mentions)
Dmi4000b, by Leica (1 mentions)
Fitc conjugated annexin 5 apoptosis detection kit, by BD (1 mentions)
Edu medium, by RiboBio (1 mentions)
Fv1000, by Olympus (1 mentions)
Fv10 asw viewer, by Olympus (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Dojindo Laboratories (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Ix83 inverted fluorescence microscope, by Olympus (1 mentions)
Edu staining proliferation kit ifluor 488, by Abcam (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Fluorescent reverse microscopy, by Nikon (1 mentions)
31 protocols using apollo 567
1
EdU Incorporation Assay for Cell Proliferation
2
Microglia-Driven NSC Proliferation and Apoptosis
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A total of 1 × 106 microglia were harvested and placed into the upper chamber of a 12-well trans-well plate (0.65 µm, Corning, New York, NY, USA), containing 1 µg/ml LPS. In addition, 1 × 106 NSCs were placed into the lower chamber, wells contained either culture medium; 40 nM leukosomes; or 40 nM lnc-EPS-leukosomes (n = 8). The microglia and medium in the control group were not untreated.
After culturing for 24 h at 37 °C, NSCs were cultured overnight using EdU medium (Ribobio, Guangzhou, China) and treated with 0.3% Triton X-100 in 1% fetal bovine serum (FBS) for 15 min at 37 °C. Then, cells were washed with PBS and to stain mitotic NSCs, 100 μL 1 × Apollo-567 (Ribobio, Guangzhou, China) was added to each well. DAPI was used to stain nuclei. Cells were evaluated and imaged using a fluorescence microscope (DMI4000B, Leica, Germany) at 565 nm and 461 nm.
The grouping and stimulation methods were identical as mentioned above. After 48 h, NSC were harvested, washed, and resuspended in PBS. A FITC-conjugated Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin, NJ, USA) was used to identify apoptotic NSCs. Procedures were strictly performed by following the kit instructions, and cell apoptosis was evaluated by FACScan laser flow cytometer (CyFlow® Cube, Partec, Germany).
After culturing for 24 h at 37 °C, NSCs were cultured overnight using EdU medium (Ribobio, Guangzhou, China) and treated with 0.3% Triton X-100 in 1% fetal bovine serum (FBS) for 15 min at 37 °C. Then, cells were washed with PBS and to stain mitotic NSCs, 100 μL 1 × Apollo-567 (Ribobio, Guangzhou, China) was added to each well. DAPI was used to stain nuclei. Cells were evaluated and imaged using a fluorescence microscope (DMI4000B, Leica, Germany) at 565 nm and 461 nm.
The grouping and stimulation methods were identical as mentioned above. After 48 h, NSC were harvested, washed, and resuspended in PBS. A FITC-conjugated Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin, NJ, USA) was used to identify apoptotic NSCs. Procedures were strictly performed by following the kit instructions, and cell apoptosis was evaluated by FACScan laser flow cytometer (CyFlow® Cube, Partec, Germany).
3
Quantitative Analysis of Cell Proliferation via EdU Assay
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EdU assays were conducted using a Cell-Light™ EdU DNA Cell Proliferation Kit (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.) according to the manufacturer's instructions. Briefly, 1×105 cells/well were seeded in 96-well plates and cultured with complete medium for 48 h at 37°C. Cells were stained with 100 µl 50 µM EdU solution for 2 h in the dark at room temperature. The cells were stained with Apollo®567 (Guangzhou RiboBio Co., Ltd.) for 30 min at 4°C and DAPI for 10 min at 4°C after cell fixation by 4% paraformaldehyde for 30 min at room temperature and permeabilization by 0.5% TRITON X-100 for 90 sec at 4°C. Three visual fields were randomly selected for observation using a laser scanning confocal microscope (FV1000; Olympus Corporation) and corresponding software (FV10-ASW Viewer; version 4.2; Olympus Corporation) at ×200 magnification.
4
Quantifying Cell Proliferation through EdU Assay
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EC cells at the logarithmic phase were seeded into a 96-well plate (4 × 104 cells/well). After treatment for 24 h, 100 μL of EdU medium was added to the cells in each well for a 2-h incubation, followed by fixation for 30 min with 100 μL of cell fixative at room temperature. Cells in each well were incubated with 2 mg/mL glycine for 5 min and with 100 μL of phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 10 min. Next, the cells in each well were stained with 100 μL of Apollo 567 (Guangzhou Ribobio Biotechnology, Guangzhou, Guangdong, China) for 30 min and 100 μL of 1× Hoechst 33342 reaction solution. After sealing in 100 μL of anti-fluorescence quenching agent, the cells were imaged under a fluorescence microscope, with the number of cells labeled with EdU counted. The EdU labeling rate (%) = [the number of EdU-positive cells/(the number of EdU-positive cells + EdU-negative cells)] × 100%.
5
Quantifying Colorectal Cancer Cell Growth and Viability
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Cell growth curve and viability were measured by CCK-8 kit (Dojindo Laboratories, Japan) as the manufacturer’s protocol. For EdU proliferation assays, cells were incubated with 50 mM EdU for 2 h at room temperature and stained with Apollo® 567 obtained from RiboBio, Guangzhou, China. The CRC cells were observed and counted with an inverted fluorescence microscope. Colony formation assays were done in line with previously reported in 6-well plates [25 (link)], 1000 cells/well. For drug sensitivity assays, cells were treated with or without oxaliplatin at indicated dose for 48 h and then suffered to flow cytometry analysis using Annexin V-FITC/PI apoptosis detection kit (Dojindo Laboratories, Japan) by a flow cytometer (BD Bioscience). All the experiments above were independently repeated for three times.
6
EdU Proliferation Assay for miR-511
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The effect of miR-511 on cell proliferation was also examined by EdU (5-Ethynyl-2′-deoxyuridine) incorporation assay. Briefly, 24 h after transfection, indicated cells were incubated with 50 μM EdU for 2 h at 37 °C according to the manufacturer’s instructions (R11053.4, Apollo 567, RiboBio, China). Cells were then washed with PBS for three times, and fixed with 4% formaldehyde for 30 min at room temperature. Next, 0.5% Triton X-100 was applied for 10 min at room temperature to permeabilize cells. After washing with PBS for three times, cells were incubated with 1 × Apollo reaction cocktail for 30 min. DNA was stained with DAPI (4′,6-diamidino-2-phenylindole) for 30 min and results were visualized with a Zeiss confocal microscope (LSM 700, USA).
7
Mollugin Inhibits Cell Proliferation
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The cells were cultured in 96-well plates, and then cells were treated with 80 μM mollugin. After 12 h incubation, the cells were labelled with 5-ethynyl-2′-deoxyuridine (EdU, RIBOBIO; R11053) for 1 h and stained with Apollo® 567 following the instructions of manufacturer. The data was measured using the Olympus IX83 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan).
8
EdU Proliferation Assay for Colorectal Cancer
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EdU assays were conducted to assess the viability of CC cells. LOVO and SW620 cells in good growth condition were seeded in a 24-well plate. The culture medium was added with the reagent from an EdU Staining Proliferation Kit (iFluor 488, Abcam Inc., Cambridge, UK) to a final concentration of 10 µmol/L, and incubated in the incubator for 2 h. The cells were then fixed with phosphate buffered saline (PBS) solution containing 4% paraformaldehyde at room temperature for 15 min and incubated with PBS containing 0.5% Triton-100 at room temperature for 20 min. Afterwards, the cells in each well were incubated for 30 min with 100 µL Apollo 567 (Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China) void of light and stained for 5 min with 4ʹ,6-diamidino-2-phenylindole. Five visual fields were randomly taken under a TCS SP8 confocal microscope (Leica, Bannockburn, IL, USA). The blue fluorescence indicates all cells, whereas the red fluorescence is the replicating cells infiltrated by EdU. The rate of EdU-positive cells was calculated.
9
EdU Assay for Cell Proliferation
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5‐Ethynyl‐2′‐deoxyuridine (EdU) assay was performed with an EdU Kit (RiboBio). C2C12 cells transfected with si‐NC or si‐MLL1 were seeded onto 12‐well plates and cultured in GM for 48 hours, and then switched into fresh DMEM medium supplemented with EdU (50 mmol/L) and incubated for 2 hours, followed by fixation, permeabilization and EdU staining with Apollo 567 (RiboBio). The cell nuclei were stained with DAPI (1:1000 in PBS). The proportion of EdU‐positive cells was determined using fluorescent reverse microscopy (Nikon).
10
Schwann Cell Proliferation Assay
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The assay of cell proliferation was performed with Cell-Light Apollo567 In Vitro Kit (RiboBio, Guangzhou, China). Complete medium was used to re-suspend the Schwann cells that were then tallied and plated on 96-well poly-L-lysine-coated plates. EdU was applied and the cells were cultured after cell transfection. The cells were fixed with phosphate buffered saline containing 4% formaldehyde and stained with Apollo 567 (RiboBio, Guangzhou, China) and Hoechst 33342 (RiboBio). Schwann cell proliferation analysis was performed using randomly selected images through a fluorescence microscope (Leica, Mannheim, Germany). The proliferating cell numbers were calculated. The average number of proliferating cells in the control group was set as 100%. The cell proliferation rate of p-GV230-Claudin-15 group was obtained by dividing by the average number of proliferating cells in the negative control or pGV230 group. The results were presented as fold change.
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