They look like dead/suffering cells. We can even see a condensed nuclei. Have you added some anti-mitogenic agent to avoid glial proliferation?(mytomicin/Ara-C?). I agree with you... they look like astrocytes (shape and size). 

Thanks Litia for the suggestion! I did try some Ara-C in the past but the neurons did not tolerate the dose.... Maybe I can try at a bit lower concentration.

Hey Huang. I've been preparing hippocampal cultures for about 10 years now, and see these circles sometimes. Is there noone in your lab with experience? They are dead cells. Either neurons or astrocytes, and both is expected for these cultures. Sometimes it will be more, sometimes less. I dont think they are harmful, and if you're imaging the cultures, I would just ignore them.

Hi Huang,

I saw similar things sometimes. You may add Ara-C and  BDNF 10ng/ml to the culture if possible. What coating you use for the culture?

Cheers,

Ke

Dear Huang,

what you see are suffering cells, one of the reasons why you see them might be related to the density of plating. If I were you, I'd try to make some small adjustments trying to plate 80%, 90%, 100%, 110%, 120% of your standard density. It is somehow the easiest of the controls you can try, since messing around with coating and medium composition would lead to an increase of the variables you already have. 

Wish you good luck!

Seba

My 2 cents. From ourexperience with parallel culturing of mice and rat neurons (same brain region), we more often see those in the rat culture than in the mouse culture. A possibility is that microglia or other cleaning process is more active in mouse culture.  ie: you get natural death in both cultures but the mouse one is cleaner. those dead cells are very sticky so do not touch them with your patch pipette, otherwise do not change anything to your protocol since the other cells look OK and you get mEPSCs.

Hope this helps.

Thank you everyone for a very informative discussion! 

@Magarethe: they actually do not affect my ephys experiment very much either. Yet I might need to do some western blot with them and am afraid that it will be bad to have those weird things there. My postdoc has quite a lot culture experience but she also did not know what they were. 

@Ke: I do not have BNDF but I might be able to find some. :) We are using Poly D Lysine for coating. 1 mg/ml.

@ Sebastian: that sound interesting! Actually since we moved to the new lab, the cell viability might have enhanced, hence leading to more cells adhering even we did not increase the plating density. I will try to reduce the density a bit to see if it helps. One other thing I might try is not flip the coverslips. (We are using wax feet to put on the coveslips and flip them up side down after plating). 

@Thomas: Ah! This might actually it. I do not really see much microglia in these recent preps and do see lots of these dead circles. How insightful. Thanks! Yet as I said above, I am worried whether these dead cells might affect biochemistry experiment and really hope to get rids of them... 

Will keep you posted! 

Cheers,Huong

Hello Huong... Higher concentrations of Poli-L-Lisine are toxic. I used to use 100ug/ml of Poli-D-Lisine and wash it very well after the incubation. How do you perform your dissociation? I do adding Tryple (1x) and pippete up and down with a glass pasteur pippete veeeeeery slowly. It is a critical step to avoid cell death.

I used to always have at least some dead neurons when doing primary neuronal culture (mouse DRG and embryonic rat midbrain). They don't really come off when feeding the cultures -- at least, not without agitating the rest of your culture enough to damage some of the neurites. Beyond keeping your cell viability as high as possible, there isn't too much you can do. 

To answer your western blot concern: I never had any bad experience with them affecting my westerns or my immunochemistry. I think this is because I would wash my coverslipped slides to remove the serum-containing culture medium -- most of the dead cells would come off during the washing process, so it wasn't a big concern.

thanks Litia and Kathleen for the advices and information! 

@Litia: Yes, too much substrate is not great. Our lab is using 1 mg/ml. This seems to be okay so far...  I do not see any typical problem of lacking or too much substrate like cells clumping or processes bundling up. We use 0.25 % Trypsin (from Worthington), incubate for 15 -17 min at 37oC. TrypLE was okay but required too long incubation, hence higher cell dead. I then titrurate with p1000 pipet and polished glass pipet. Yes, you are definitely right that the dissociation is super crucial. Last week I made the glass pipette a bit smaller and dissociated the cells a bit harder. They did not at all like it. The counting was not different but the cells that ended up adhering to the coverslips are just not very happy. Slow maturation. Sparsely looking. I dissociated them much gentler this time... 

@Kathleen. Hah, that is a realy good point that the cells dead should be falling off. The weird thing is that these guys seem to not at all falling off though. They are still there when I move coverslips from one solution to another. Did you look at the coverslip after washing and see that those dead cells were gone? Do your dead cells look like the ones in my picture? My concern is if these things are not actually dead cells? When I looked at them with 40X objectives, some of them have very odd processes... (images attached, I took these photos while recording from neurons). Please let me know if you have any suggestion

Hello Huong, I've recently come about this problem in my hippocampal and cortical cultures in mice at E17.5. Since I'm taking RNA from these I'm afraid these will affect my experiments. Were you able to solve this issue? I tried washing with PBS ~3 hours after plating and before changing media but they don't seem to detach easily. I've also noticed that they last a very long time in culture without entirely degrading. 

Hi Angelica, I haven't completely solved the issues but found a few factors that affected the appearance of these round circles. Just to clarify, we are doing Banker style culture, with an astrocyte feeder layer in the bottom and neurons plated on glass coverslips facing the astrocytes. When the astrocytes are too fresh, or when we had bad B27 lot numbers that promote growth of non-neuronal cells, there were way more dead neurons/circles thingy. We had tried to lower the density of the astrocytes feeder layer, and also tried different B27 lots to minimize the issue. Another factor that contributed is the distance between the astrocyte layer and the coverslip with neurons. I learnt that if there was not enough distance between the two, there were more dead cells. So, I just simply made bigger wax feet to increase the distance. Hope this helps!

Huong