I´ve been working with BV2 cells for a little over a year now and am recently trying to work with the OGD model. I tried out different incubation times & amounts of oxygen (1 % O2 for 6 h, 0 % O2 for 3 h) and measured fitness via MTS-Assay, nitrite in supernatants via Griess-Assay and cytokine expression via RT qPCR. And even though I see a decreased fitness I never see any NO production. The expression of TNFa & IL-1b was even lower than in my control which I thought could be because I first used SILAC medium for the OGD, that´s why I switched to using DMEM now. This lead me to the next problem: now after 3 h with GD and 0 % O2 almost all my cells die - only 2 % survived. I tried out shorter OGD (1 or 2 h with 0 % O2) but the viability still seems very low.

Since the OGD model for BV2 cells is very well established (even with harsher conditions!), I went looking for solutions and found out that in some of these papers the cells had a more elongated form. My cells are amoeboid and stay that way during the experiments, I will attach a picture.

That leads me back to my question: do any of you know, if (and if yes, how) the morphology of the cells influences the outcome of my experiments? Or do you have any other recommendations for making the OGD model work? I already read other threads about BV2 cells but couldn´t find a solution.

I cultivate my cells in DMEM/F12+10 % FCS and never experienced something other than amoeboid like cells (except for a few elongations after LPS-treatment). For the OGD experiments I use glucose free DMEM + HEPES & GlutaMax and add supplements to the control (sodium-pyruvate, glucose, 10 % FCS).

Thank you for your help! :)

PS: you can see the different morphology in this thread: https://www.researchgate.net/post/Why_am_I_always_the_terminator_of_BV-2_cells