Hi Laura,

I also work with primary murine glial cells and I know what kind of clumps do you see. I don't know any specific way of getting out of them but I have two suggestions that maybe could help you:

- Seeding less number of cells: I guess you are using an optimized cell density in your PDL coated flask but maybe by decreasing the cell density you reach less clump formation when you homogenate the resuspended cells

- Resuspend cells using a narrow pippete (5 ml one) or also by using pipette tips (maybe you can take a litlle volume of your resuspended cells and make it pass through a 200 ul tip. You'll see that you can break mostly all this clumps). In my hands making the resuspended cells passing through a 1000 ul or a 200 ul pippete tip don't make any harm to the cells and they dissociate in a better way

Hope it helps

Ángel

You could also consider passing your cell suspension through a 40 or 70 um filter/cell strainer. Beyond excluding the clumps, it should also help dissociate them.

Hi Laura,

You can also try the seeding with a P1000 automatic pipette fitted with fire-smoothed sterile filter tips. It is a reliable method in our hands when we want to dissociate cell clumps (we usually do it with neurospheres).

Hi Laura,

Attached is our protocol for astrocytes culture. It works well for our practice. If your isolation protocol works well, you should get very good monolayer of astrocytes. We always use Accutase to dissociate astrocytes and can get single cell without clumps. Let me know if you have more questions.

Isolation protocol for mouse/rat astrocytes culture

Coating

1. Coat plates with Poly-L-Iysine (1mg/ml) for 4 hours at RT [ 4oC one day]

2. Wash plates with dH20 (2-3 times) [autoclaved and filtered]

3. After dry, add astrocytes culture medium (DMEM/F12, containing 10% FBS, 1% glutamine and Antibiotic), leave in 37oC incubator, ready for cell plating.

Dissection/processing of tissues

1. Do dissection (P2, 2-3 pups) in Earle's Balanced salt solution (EBSS - Gibco 0766) + Hepes (1: 100 from 1M stock (5ml))

2. Transfer tissues [cut mouse/rat cortical tissue into smaller pieces] to enzyme solution (10ml EBSS with 10mM HEPES + 200ul papain (20-25 units/ml final solution strength) - sterilized solution by filter. Incubate at 37oC for 30 mins.

3. Transfer to new tube, wash with DMEM/F12, pipette a few times to dissociate cells.[in about 15-20 ml DMEM/F12]

4. Pass through cell strainer. [filter in 50 ml falcon and split to 15 ml tubes- 3-5 ml each]

5. carefully add 7.5% BSA in PBS (0.5ml) to the bottom of the tube.

6. Spin @ 1800 rpm for 5 mins

7. first remove the interface, then the media [ important]

8. distribute about 1ml medium to all the tubes to resuspend pellets and then transfer to one tube

9. Plate the cells in astrocytes culture medium (DMEM/F12, containing 10% FBS, 1% glutamine and Antibiotic)

I assume you're trying to get single astrocytes from the start of the dissection protocol. You can't. You need to grow the astrocytes for a couple of weeks first and then seed them as single cells. As Dongliang said before, you need to get a monolayer first.

During the extraction of cells from the brains, you get a lot of debris that will stick to cells and form clumps. This is normal. So, seed your cells, let them grow and when the plate is confluent and clean you can trypsynise them (you can also try EDTA 5mM in PBS for 5 min).

You can reduce the debris and clumps by using cell strainers (70 um) and DNAse, but you can't avoid them.

Also, I think it should be better to get the astrocytes from a specific part of the brain (such as cortex) instead of the whole brain. That way the type of astrocytes that you get at the end are similar. And don't forget to remove meninges when dissecting your samples, or you'll get a lot of fibroblasts.

Hello Dongliang Ma , thanks for your help! I still get astrocytes cell clumps even though I am using cell strainers. Now I am going to try to dissociate my astrocytes with your protocol. Can you tell me the accutase concentration you are using? Thanks in advance! Greetings, Laura

Hi Laura,

I suspect you try to use the cells for your experiments from isolation directly. The cells from isolation are the mixed population (could not get pure astrocyte) including immature neuron, neural progenitor cells and glia ... In this case, when you seed and culture the cells from isolation with astrocyte medium (with FBS) for selection, you will kill most of neural cells. After 1-2 passages, you could get relatively pure astrocyte for your experiment. I assume your cell clumps are from neural progenitor cells.

By the way, Accutase, Sigma, #A6964 is ready to use.

We use papain for isolation and accutase for next splitting the cells as i mentioned in the above protocol.

Hope it helps!

Hi Dongliang Ma ,

I am not trying to use the cells directly from isolation. In fact, I am isolating the astrocytes after harvesting the microglia up to 4 times. Unfortunately, I am still having issues getting single astrocyte cells. Do you have any clue why there are sticking together forming these big chunks of cell clumps?

Thanks in advance!

Greetings, Laura