Hello,
I have been finding different protocols for culturing murine BMDCs. Researchers sometimes use different plates for culturing them (i.e. Petri dishes, non-TC treated, TC grade treated; 6-well plates; 24-well plates;...).
My aim is to obtain the highest percentage of dendritic cells, and so the less amount possible of monocytes/macrophages.
Which kind of plate would you recommend, and why?
At which concentration should the bone marrow cells be seeded in the type of plate you are suggesting?
In which type of plate, and at which concentration, should the BMDCs be re-seeded once differentiated?
Thank you very much in advance.
Francesco
Hi Francesco,
In my lab we usually have used Petri dishes (the same used to cultivate bacteria) during the differentiation (about 10 days). We seed 10e7 cells/dish in 7 ml medium, using RPMI 1640 containing 10% serum 1% pen/strep and supplemented with recombinant murine GM-CSF (1000U/ml, peprotech). Considering that the seeding day is the day 0, we add more 5 ml of the complete medium described above on day 3 and on the days 5 and 7 we put the dishes inside a cabinet and wait 15-20 min to the cells to settle, gently aspirate 4 ml of each dish and 5 ml of a fresh medium. On the day 10 we gently collect the cells (using PBS, 2x 5mL), centrifugate for 10min 1200 rpm 4ºC and count it. We usually collect 20% of the cells seeded on day 0. I use the following formula: each 10 Petri dishes with 10e7 cells will give me 1 24-well plate with 5 x10e5 cells/well. I know, is not much but at least the % of DCs is very good.
Finally we seed the cells in 24-well plates with 5 x10e5 cells/well (day 10) using a fresh medium without GM-CSF and estimulate cells on the next day.
I hope it helps.
Priscila
I forgot to say that our RPMI is supplemented with 1% Hepes.
You're welcome!
Hi Priscilla, sorry for the extreme delay, and thank you for adding this detail.
We put 0.5% Hepes; would you say is too low?
Regards
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