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Views: 21 Author: vivi Publish Time: 2025-05-09 Origin: Site
To compare the culture performance (proliferation and apoptosis) of the C1-191L air-jacketed CO2 incubator and a certain brand of CO2 cell incubator across different cell types.
Proliferation Assays: BrdU, CFSE, eFluor 450
Apoptosis Assay: Annexin V/7-AAD cell apoptosis detection kit
BrdU (5-Bromo-2'-deoxyuridine) is a synthetic analog of the DNA precursor thymidine. During the S phase of the cell cycle, BrdU can replace thymidine and selectively incorporate into newly synthesized DNA. When cells are in the DNA synthesis phase and BrdU is present in the environment, it is inserted into replicating DNA strands either via in vivo injection or cell culture. This substitution is stable and can be passed on to daughter cells. As long as the cells remain viable, BrdU remains in the nuclear DNA over a long period. The incorporated BrdU can be detected through methods such as flow cytometry or absorbance detection using a microplate reader, allowing researchers to assess the cell proliferation capacity.
1) Harvest mouse spleen to prepare a single-cell suspension, sort CD4+ T cells (the sorting procedure for primary cells has been described previously and is omitted here). Count the cells, and seed 1×10⁷ cells per well in a 12-well plate (1 mL/well). Pre-incubate the plate in the incubator (37°C, 5% CO2).
2) After 48 hours of culture, add 5 µL of BrdU (final concentration: 10 µM) per 1 mL of medium and mix gently.
3) Incubate the plate in the incubator for 4–5 hours, then harvest the cells.
4)Stain surface antigens, followed by intracellular BrdU staining using the BrdU antibody (according to the kit instructions).
5)Analyze using flow cytometry, process the data with FlowJo software, and plot the results.
CFSE (Carboxyfluorescein diacetate succinimidyl ester) is a cell-permeable fluorescent dye that contains both a succinimidyl ester group, which specifically binds to cellular proteins, and acetate groups that enable passive diffusion across the cell membrane. In its diacetate succinimidyl ester form, CFSE is non-fluorescent but membrane-permeable, allowing it to enter cells freely. Once inside the cell, intracellular esterases hydrolyze the acetate groups, converting CFSE into a highly fluorescent and membrane-impermeable form.
The active CFSE molecule then covalently binds to intracellular proteins, particularly the free amine groups of cytoskeletal proteins, forming stable fluorescent conjugates. As the cell divides, the fluorescently labeled cytoplasmic proteins are evenly distributed to daughter cells. With each cell division, the fluorescence intensity is halved—second-generation cells exhibit half the fluorescence of the parent generation, and third-generation cells display half of that of the second, and so on.
This reduction in fluorescence intensity can be detected and analyzed using flow cytometry under 488 nm excitation light. By monitoring the progressive decline in fluorescence, the proliferation and division rate of the cells can be quantitatively assessed.
CFSE Staining Diagram
1) Harvest mouse spleen to prepare a single-cell suspension, sort CD8⁺T cells, count the cells, and adjust to a concentration of 1×10⁷ cells/mL.
2) Prepare CFSE working solution at a final concentration of 10 µM. Mix 1 mL of CFSE dilution with 1 mL of the cell suspension and incubate at 37oC in a water bath for 10 minutes.
3) Add 5–10 mL of culture medium containing 10% serum to stop the staining, centrifuge, and wash. Repeat the washing step three times. For the final wash, incubate the cell suspension at 37oC in a water bath for another 10 minutes, then centrifuge.
4) After centrifugation, resuspend the cells in culture medium, count, and seed them into a 96-well plate at the desired density (200 µL/well). Incubate the plate in the CO2 incubator (37oC, 5% CO₂).
5) After 5 days of culture, collect the cells.
6) Analyze using flow cytometry with excitation at 488 nm.
7) Use FlowJo flow cytometry analysis software to analyze the data and generate plots.
3.2.3 Experimental Results
Cell Proliferation Dye eFluorTM 450 is a violet fluorescent dye used to monitor individual cell divisions. This dye covalently binds to cellular proteins that contain primary amines. As the labeled cells divide, the dye is evenly distributed between daughter cells, resulting in a halving of fluorescence intensity with each cell division. By analyzing the successive halving of fluorescence, the proliferation rate of cells can be accurately assessed.
1)Harvest mouse spleen, prepare a single-cell suspension, sort B cells, count the cells, and adjust to a concentration of 1×10⁷ cells/mL.
2)Dilute eFluor 450 dye to a final concentration of 10 µM. Mix 500 µL of the diluted eFluor 450 solution with 1 mL of the cell suspension and incubate in a 37oC water bath for 15 minutes, gently mixing every 3 minutes.
3)Add pre-warmed complete culture medium to terminate staining, then centrifuge.
4)After centrifugation, wash the cells once with 37oC pre-warmed PBS, centrifuge again, and discard the supernatant.
5)Resuspend the cells in complete culture medium, count them, and seed into a 96-well plate at the desired density (200 µL/well). Incubate at 37oC with 5% CO2.
6)After 5 days of culture, collect the cells.
7)Analyze the cells using flow cytometry, using the DAPI channel (excitation at 405 nm).
8)Analyze results and generate graphs using FlowJo software.
Primary mouse CD4+ T cells, CD8+ Tcells, and B cells were cultured simultaneously in two different CO2 incubators under identical conditions. Cell proliferation was assessed using flow cytometry. Results from the BrdU and CFSE assays indicated no significant difference in the proliferation of CD4+ and CD8+ T cells between the two incubators. However, data from the eFluor 450 assay showed that B cell proliferation varied between the two incubators, with the CI-191L air-jacketed CO2 incubator demonstrating slightly better performance in supporting B cell growth.
