I have to treat the BV2 cells repeatedly for 5 days (daily treatment) with an immuno-modulator. However, the problem is that BV2 morphology changed after 72 hr and they start to die after 4 days. Could you please suggest the optimum seeding concentration of BV2 cells so that they can sustain for 5 days in healthy morphology? Or can anybody recommend the publication that contains the repeated treatment (2 or 3 times a day) for short period of time?
The optimum concentration must be fixed as in the recommended method but i think it is better to think how could you keep this cell active during this long term of treatment by adding supplements that help keeping the cell health and live
as some essential amino acids or fetal bovine serm or vitamins.
Having worked on BV2 myself, I can assure you that it is rather impossible to keep these cells for 5 days healthily in culture. These cells tend to get activated in stress and start to float after a while. The problem is if you grow these cells in lesser densities, they tend to proliferate and aggregate and kinda form 'colony' of cells and are not uniformly spread, and this again causes them to float once they are 2-3 days in culture. A very high density, of course is not possible given that they proliferate quite fast (if the passage number is high). Can you please specify the passage number (if available), or the % FBS used for culturing these cells? In addition, for me to give you an idea of the optimum cell densities, I would like to know the culture dishes you are growing them in.
Hi, I use BV-2 cells for 4-days culture and it works OK. I seed them around 20 000 cells per well on 96 well plate. I think for 5 days you could try even lower concentration for seeding. The medium should keep the same color after 24 h incubation (if it is too yellow, than you probably have too many cells).
It is important that you change the medium inside the wells every day when you make the treatment. More, the volume of the medium will also affect the condition. I add 200 microliter per well on 96-well plates (when I used 100 microliter per well than it turned too yellow after 24 h). Also, I use 2% FBS in the medium - that makes them growing slower (but they are still OK with this FBS concentration). I use 10% FBS medium for culturing in the flask and for seeding on the multiwell plate and later treatments I use 2% FBS.
Also, as BV-2 cells are semi-adherent it is better to use a plate with small wells (such as 96-well). I tried to culture them on 6-well plate and then it was diffcult to have them confluent (a lot of them detached). I hope it will help, good luck
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