I am doing a one-day assay with primary monocytes and seeding them at 3*10^5/well, as per a colleague recommendation. However I see others recommend much lower, but they seem to have different experimental goals (time-course, proliferation etc). Are there any reasons I might be seeding too high? I need a lot of RNA for qpcr after the assay.
use common rules:
1. density no more than 70 % of monolayer (cell number will vary of cell size, primary cell usually much smaller than cells from strain)
2. final concentration no more than 1x106 cell/ml of media (better to use at least twice lower ;)
We've successfully used peritoneal macrophages in concentration up to 200000 per well (96 well plate), but non-linear response was observed at maximum concentration (LPS and Zymosan response). So, depending of your aim (experimental conditions) try to optimize cell concentration or take other plate (48 or 24 wells).
Test serial dilutions of cells to identify optimum. I guess you will find it in range 10000-50000 cell per well.
Alternatively use column method of RNA isolation to maximize yield.
From Rajendra's link:
"In general, at least 1 x 10^5 cells/cm2 can be produced when growing cells as attached monolayers in culture. The average cell yields used here are based on this number. Actual cell yields can easily be several times higher than this depending on the cell line and culture conditions"
so if I switch to 48-well size I should be able to seed my 3*10^5 cells per well so I have enough RNA for qPCR (many genes). Decreasing cell numbers is not an option; I won't have enough RNA.
If I remember right, I was doing a 3 day proliferation experiment and did a "titration" to determine optimal cell number for a 96well plate using primary BMDMs.
I think I ended up starting the experiment with 2,000-5,000 cell/well? I think I may actually have the pictures from the titration I did (if you want to get in touch via email, maybe if you still cant get it to work right?).
BOTTOM LINE: everyone (else besides D.V.--200,000 is crazy, I think it's a typo) has pretty sound advice probabaly around 20,000 - 30,000.
But honestly I like Justines idea the best!!!
and switch (if you can) to two 48well plates or maybe even 4 24well plates!
Side note:
The 70% confluence standard in commly used accross a variety of macrophage assays. The experiment I know for sure that 70% is well accepted is: cytokine stimulation western blots experiments.
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