I greatly appreciate all of your help! I've finally finished my BV-2 experiments and got a paper published. Here are some of my experiences on BV-2 culture.

1. You should pay attention to the original medium which BV-2 cells are kept in when you receive the cells.

I have received a gift BV-2 from my friends' lab (kept in 1640 medium) at first and after the cells have all gone, I bought  another copy (kept in MEM medium) from a bio-company. But my problem is that I never care about the original medium the cells were kept in before. I just listened to the senior students and used the DMEM with high glucose according to the experience in my lab. At last, I realized it and asked my friend again for another cell block and kept the cells in the same medium, it turns out that the cells grew well.

In my opinion, as consistent with what Lindsay said, BV-2 cells may get a better condition under the basal culture medium of DMEM/F12 or 1640.

2. DO NOT subculture BV-2 cells until they go confluent.

BV-2 cells are a group of rapidly dividing immortalized cells. Their proliferation speed is amazing. Thus just as what Sun Hongbin indicated, I should have subcultured the cells when they were in the first picture. This is exactly how I promoted my culture protocol: subculture the cells when the density is about 80%-90% of the 100 cm2 dish. Don't wait any longer.

3. DO NOT trypsinize BV-2 cells excessively.

According to my experience, you can use 2-3 ml preheated 0.25% trypsin to add to the 100 cm2 dish. After gently waggling the dish to make trypsin cover the subface for 5 seconds, the trypsinization can be terminated. Otherwise, if the period of trypsinization is longer, the cells may be excessively trypsinzed and injured. It may influence the cellular condition thereafter.

These are what I have concluded after dealing with BV-2 cells for nearly 4 months. And I am so glad to share them with everyone!

Thanks again for suggestions from Ernesto Alfaro-Moreno, Tomas Machacek, Lindsay Spielman and Sun Hongbin. O(∩_∩)O~

Similar question previously on Researchgate. This might help you: https://www.researchgate.net/post/Can_anyone_advise_me_on_how_to_maintain_BV_2_cell_line

Have you tried to add glutamine to the medium? It is pretty essential for energy metabolism as well as nucleic acids and protein synthesis. Cells can usually prepare it themselves (by glutaminesynthetase) but it costs ATP.

In my experience using BV-2 cells, I have found that they are very high maintenance.  Once the cells are grown up from stock they require fresh media on a constant basis.  In our lab we passage the cells every other day, or there's a high chance of cell death (meaning someone has to come in on the weekends to maintain them). We use DMEM F12 media with 10% Calf Bovine Serum and 1% penicillin / streptomycin / amphotericin B. Hope this helps. 

 In my view, BV2 in the first photo  is cultured well, and it should be subcultured. If you don't passage, the cells in second photo are your results. Besides, you can  simply isolate them down with fresh medium. Once the concentration was very high, BV2 will be clustered, you can use 0.25% trypsin .

I greatly appreciate all of your help! I've finally finished my BV-2 experiments and got a paper published. Here are some of my experiences on BV-2 culture.

1. You should pay attention to the original medium which BV-2 cells are kept in when you receive the cells.

I have received a gift BV-2 from my friends' lab (kept in 1640 medium) at first and after the cells have all gone, I bought  another copy (kept in MEM medium) from a bio-company. But my problem is that I never care about the original medium the cells were kept in before. I just listened to the senior students and used the DMEM with high glucose according to the experience in my lab. At last, I realized it and asked my friend again for another cell block and kept the cells in the same medium, it turns out that the cells grew well.

In my opinion, as consistent with what Lindsay said, BV-2 cells may get a better condition under the basal culture medium of DMEM/F12 or 1640.

2. DO NOT subculture BV-2 cells until they go confluent.

BV-2 cells are a group of rapidly dividing immortalized cells. Their proliferation speed is amazing. Thus just as what Sun Hongbin indicated, I should have subcultured the cells when they were in the first picture. This is exactly how I promoted my culture protocol: subculture the cells when the density is about 80%-90% of the 100 cm2 dish. Don't wait any longer.

3. DO NOT trypsinize BV-2 cells excessively.

According to my experience, you can use 2-3 ml preheated 0.25% trypsin to add to the 100 cm2 dish. After gently waggling the dish to make trypsin cover the subface for 5 seconds, the trypsinization can be terminated. Otherwise, if the period of trypsinization is longer, the cells may be excessively trypsinzed and injured. It may influence the cellular condition thereafter.

These are what I have concluded after dealing with BV-2 cells for nearly 4 months. And I am so glad to share them with everyone!

Thanks again for suggestions from Ernesto Alfaro-Moreno, Tomas Machacek, Lindsay Spielman and Sun Hongbin. O(∩_∩)O~

Dear all, 

I am beginning to work this microglial cell line, so I really appreciate all your comments. I would like to know if you recommend using any speed to pellet them down. I guess 1000-1500 rpm 5 minutes is fine for them.

Many thanks in advance!

Dear Anna,

According to my lab, 1000-1200 rpm for 5-8 min is fine for almost all kinds of cells. But I usually skip the centrifugation step.

I just wagged the dish to make trypsin cover the dish subface for 5 seconds and then trypsin was pipetted and discarded. About 2 ml culture medium was added and the cells were blew off the dish subface by a pipette. You can see the cells falling down in patches directly. Then you can transfer them into a centrifuge tube and disperse the cells using a pipette or you can simply disperse the cells in the dish. After that, you can subculture the cells into another 4 or 6 dishes.

You can get much more knowledge while trying! Good luck!

 Dear all,

I am facing a similar problem to the one was discussed here and I would highly appreciate your help since the only BV-2 cells I have are the ones in culture and I bought them to ICLC. I would rather recover the ones I have now than buying a new vial, if possible.

Two days ago I thawed the only BV-2 microglia vial in RPMI media since it was frozen in that media. 24 hours later I realized that around 30% of the cells were not attached, and I centrifuged the supernatant and I seeded the cells in DMEM, RPMI and DMEM:F12. From the cells I collected from the supernatant, I realized that there are more attached cells in DMEM or DMEM:F12 compared to RPMI media condition. I am not sure if this may be due to the fact that RPMI ise also used when cells are grown in suspension (and therefore those cells are viable), or which the cause might be. Is that normal to have cells in suspension in BV-2 cultures? Or should I be worried about? Can it be an inflammation?

I kept the attached cells in RPMI media and the vast majority of them are still attached.

My main concerned is the fact that the vast majority of my different plates (attached cells ffrom the very beginning, supernatant maintained in DMEM, DMEM:F12 and RPMI) look like the second picture of this post (so without elongations). I would like to know if there is any way to recover these cells or if it takes some days for the cells to get those elongations. Do you think is worth doing adding primocin or plasmocin to the media so I could get rid of any potential micoplasma contamination that could avoid my cells to get protrusions?

Many thanks for your help!