EdU assay was used to examine cell proliferation. Glioma cell lines in the logarithmic growth phase were seeded into 96-well plates at a density of 2×10³- 4×10⁴. After 24 h, the adherent cells to the wells were transfected. Five parallel wells were set up for each group. Cells in each well after transfection for 48 h were cultured with 100 μL EdU medium (RIBOBIO, China) for 2 h and fixed with 100 μL of cell fixation solution (PBS containing 4% polyformaldehyde) for 30 min at room temperature. Subsequently, the cells were incubated with 2 mg/mL glycine (Solarbio, China) for 5 min, rinsed with 100 μL of PBS containing 0.5% TritonX-100 (RIBOBIO, China) for 10 min, and stained using 1 × Apollo staining reaction solution (RIBOBIO, China) for 30 min in conditions devoid of light. Next, the cells were reacted with 100 μL of the 1 × Hoechst 33342 reaction solution (RIBOBIO, China) for 30 min and sealed with 100 μL of the anti-fluorescence quenching agent. Six to ten fields of view were randomly selected for each well and photographed under a fluorescence microscope.
Apollo staining reaction solution
Manufactured by RiboBio
Sourced in China
The Apollo staining reaction solution is a laboratory reagent used in various staining processes. It is a concentrated solution that can be diluted and applied to samples for the purpose of staining. The core function of this product is to provide a chemical reaction that enables the visualization of specific cellular or molecular components within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342 reaction solution, by RiboBio (4 mentions)
Edu medium, by RiboBio (3 mentions)
Tritonx 100, by RiboBio (2 mentions)
Glycine, by Solarbio (2 mentions)
Dm4000 microscope, by Leica (2 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
6 protocols using apollo staining reaction solution
1
Quantifying Glioma Cell Proliferation
2
Quantifying Cellular Proliferation via EdU Assay
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EdU assay was used to examine cell proliferation. Glioma cell lines in the logarithmic growth phase were seeded into 96-well plates at a density of 2×10³-4×10⁴. After 24 h, the adherent cells to the wells were transfected. Five parallel wells were set up for each group. Cells in each well after transfection for 48 h were cultured with 100 μL EdU medium (RIBOBIO, China) for 2 h and xed with 100 μL of cell xation solution (PBS containing 4% polyformaldehyde) for 30 min at room temperature. Subsequently, the cells were incubated with 2 mg/mL glycine (Solarbio, China) for 5 min, rinsed with 100 μL of PBS containing 0.5% TritonX-100 (RIBOBIO, China) for 10 min, and stained using 1 × Apollo staining reaction solution (RIBOBIO, China) for 30 min in conditions devoid of light. Next, the cells were reacted with 100 μL of the 1 × Hoechst 33342 reaction solution (RIBOBIO, China) for 30 min and sealed with 100 μL of the antiuorescence quenching agent. Six to ten elds of view were randomly selected for each well and photographed under a uorescence microscope [9] .
3
Proliferation Assay for CD24(+) and CD24(-) SCAPs
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The fifth generation CD24 (+)-SCAPs and CD24 (−)-SCAPs were prepared with good growth condition. After digested with 0.25% trypsin, 100 ul cells (4 × 104/mL) were incubated in a 96-well palatefor 4 h at 37 °C in 5% CO2. Each group of cells were reconstituted in 3 wells. After discarded the culture medium, 150 μL of EdU medium (containing 25 μmol EdU, 10% FBS α-MEM, Hyclone) was added to each well of the cells. After incubating for 48 h at 37 °C in 5% CO2, the cells were washed twice with PBS for 5 min, and 50 μL of 4% paraformaldehyde was added to each well and the cells were fixed for 30 min at room temperature. Then, added 50 μL 2 mg/ml glycine to the cells and shaken for 5 min, and, added 100 μL of penetrant (0.5% Triton X-100) to each well and shaken for 10 min. After washed the cells with PBS for 5 min, 100 μL Apollo staining reaction solution (Ribobio, Guangzhou, China) were added to each well and shaken for 30 min in the dark. After washed 3 times with formaldehyde solution, 100 μL Hoechst 33,342 reaction solution (Ribobio) was added to each well and incubated for 30 min at room temperature. Then washed the cells 5 times, and added 100 μL PBS to each well for observing under an inverted phase-contrast microscope (Olympus).
4
BMSC Transfection and Proliferation Assay
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BMSCs transfected with miR-NC inhibitor or miR-155 inhibitor were seeded in 6-well plates (1×104 cells/well) and cultured with or without 50 mM puerarin for 7 days. Images were captured using a Leica DM 4000 microscope at a magnification of ×100 (Leica Microsystems GmbH) and cell viability was determined by refractive index at 450 nm using a Bio-Rad 680 spectrophotometric microplate reader (Bio-Rad Laboratories, Inc.). To determine BMSC proliferation, cells were fixed with 4% (v/v) paraformaldehyde for 30 min at room temperature, and then stained with 100 μl Apollo® staining reaction solution (RiboBio) for 15 min at room temperature. The BMSCs were incubated with Triton X-100 (0.5%) for 5 min and stained with 1X Hoechst reaction solution (500 μl). After three washes in PBS, images were captured using a fluorescence microscope at a magnification of ×100 (Olympus Corporation). The percentage of EdU-labeled cells was calculated from at least six randomly selected fields in each well. For differentiation, BMSCs were cultured for 21 days and subjected to Oil Red O staining. Cells were washed with PBS three times and images were captured at a magnification of ×100 with a Leica DM 4000 microscope.
5
Quantitative Cell Proliferation Assay with EdU
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An EdU assay was used to detect cell proliferation. A total of 2×105 THCA cells/well (HTH-83, K-1, or TPC1) were plated in 96-well plates. After 24 h, the adherent cells were transfected. After 48 h, 100 µl EdU medium containing 10 µM (Guangzhou RiboBio Co., Ltd.) was added to each well for 2 h, and the culture medium was washed and fixed with 100 µl cell fixator (cat. no. P1110; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 30 min. The fixed cells were permeabilized with 100 µl PBS containing 0.5% Triton X-100, followed by staining using an Apollo staining reaction solution (Guangzhou RiboBio Co., Ltd.) in the dark for 30 min at 37°C. A Hoechst 33342 reaction solution (100 µl 1×; Guangzhou RiboBio Co., Ltd.) was used for 10 min at 37°C. The dyed plate was placed under an inverted fluorescence microscope (×100 magnification) to obtain fluorescence images.
6
EdU Incorporation Assay for PSCs
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PSCs were incubated with EdU-containing medium for 2 h, then, fixed with 4% paraformaldehyde for 30 min, washed with PBS, incubated with 2 mg/mL glycine for 5 min, washed with PBS for 5 min, incubated with 0.5% TritonX-100 for 10 min, then incubated with Apollo staining reaction solution (Ribobio, China) for 30 min, washed with PBS and stained with DAPI reaction solution for 10 min. Observed with a fluorescence microscope.
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