I am using primary bonemarow derived macrophages and in the assay kit data sheet they stated that the typical seeding for the cells is between 2500-100000
1- ( if anyone did this could you please recommend the proper cell number?).
2- typical incubation time stated is between 1-72h.
( could you please suggest the proper incubation time with the Brdu?
Many thanks
Hi Jabbar,
We typically perform this assay with EdU, but the incorporation kinetics are the same regardless of probe. BrdU incubation time varies depending on what you hope to measure. A 2 hour incubation is useful to determine % of cells in S-phase of the cell cycle. Longer incubations (24+ hours) will give you a better idea of the percent of cells that are proliferating.
Plating density varies of course depending on the growth vessel. For a 96-well plate we typically use 7.5-10k/well depending on how long cells will be grown.
Chris
Hi Jabbar
In Our lab, We routinely use BrdU Assay kit from cell signaling. Typically the seeding density and BrdU incubation time depend on the cell morphology (former case) and doubling time (I assume it is 20h for primary bone marrow-derived macrophages)
I perform this assay most of the time using fibroblast isolated from human lung primary cultures. the seeding density I use is around 10k/well when using 96 well plate and typical BrdU incubation time is around 24hrs after the treatment period (if using inhibitor or inducer of proliferation). I Hope this will help you to some extend to figure out your conditions.
Good Luck!
Hi Jabbar,
We typically perform this assay with EdU, but the incorporation kinetics are the same regardless of probe. BrdU incubation time varies depending on what you hope to measure. A 2 hour incubation is useful to determine % of cells in S-phase of the cell cycle. Longer incubations (24+ hours) will give you a better idea of the percent of cells that are proliferating.
Plating density varies of course depending on the growth vessel. For a 96-well plate we typically use 7.5-10k/well depending on how long cells will be grown.
Chris
Hi Sontake
Thank you very much for your help. It is very much appreciated
Hi Chris
Thank you very much for your help. I fellowed your recommendations and Sontake. That was a great help.
Many thanks
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