To check the percentage of S-phase cells in the cell cycle, cells from EP, MP and LP were trypsinized from gels and TCP and were seeded on collagen-coated glass coverslips as described above. After 48 h of seeding, BrdU reagent (Invitrogen, 000103) was added in 1:100 (v/v) ratio in media and incubated for 4 h at 37°C in a humidified incubator with 5% CO2. Thereafter cells were fixed (4% paraformaldehyde), permeabilized (0.5% Triton-X), denatured (2 M HCl), blocked (1.5% bovine serum albumin), and incubated with anti-BrdU antibody (Invitrogen, B35128, 1:100) and counterstained with AlexaFluor 568 (Invitrogen, A11061, 1:400). Immunofluorescence images were captured using EVOS-FL auto and BrdU positive and negative cells were counted manually using ImageJ.
Brdu reagent
Manufactured by Thermo Fisher Scientific
The BrdU reagent is a laboratory product that can be used to detect and measure cell proliferation. It functions by incorporating into DNA during the S-phase of the cell cycle, enabling the identification of cells that are actively dividing.
Automatically generated - may contain errors
2 protocols using brdu reagent
1
Cell Cycle Analysis via BrdU Incorporation
2
Cell Proliferation Quantification via BrdU
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48 hrs post-seeding, cells were incubated with BrdU reagent (Invitrogen, 1:100) for 4 hrs at 37°C with 5% CO2. This was followed by fixing (4% PFA), permeabilizing (0.5% Triton-X), denaturing (2M HCl), blocking (1.5% BSA), and incubating with primary antibody (Invitrogen, B35128, 1:100). Images were captured by EVOS-FL Auto and analysed with ImageJ.
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