I am trying to see an interaction between a protein secreted by astrocytes with microglial surface receptor. During the glial culture itself, I will be separating microglia from astrocytes by shaking so I remove them on around Day 7. I wanted to ask if it is possible to co-culture them again after this point? Since, the protein I am interested is secreted into the media, I believe transfer of media could work as well. But I am not sure about the steps to follow in order to do so.
Hi Punam
There are indeed several ways of doing this. The first is, as you describe, simply to treat the separated microglia with media conditioned by the astrocytes. In this case, you would seed the microglia in a plate (you may need to coat with PDL) of appropriate size (depending on your experiment) and culture them overnight. On the following day aspirate their media and replace it with media conditioned by an astrocyte culture. Depending on what type of samples/measurements you wish to take you will then incubate the cells for an appropriate length of time before harvesting or performing your assay.
Another potential approach would be to seed the astrocytes into plates and culture microglia on transwell inserts suspended above the astrocyte layer. This is a non-contact co-culture which can be maintained for at least several days prior to your assay.
The most appropriate method will depend on the specifics of your experiment. The transwell method normally works well for diffusible factors, but one potential flaw is that inserts tend to be rather small so if you require lots of cells/protein per sample you might struggle. Timescale is another factor to consider - if the cells only need to be exposed to the factor briefly then media transfer is probably the way to go, but if prolonged exposure (several days) is required then transwells might provide a better solution as the cells will be constantly supplied with freshly produced factors.
Can you give any more details of what exactly you want to do?
To James Cook
Hi James. Thank you for your reply. I am trying to look at an interaction between a microglial receptor (siglec) and a sialylated proteins released by astrocytes to see how it changes microglial acitivity and morphology?
Hi Punam
So it sounds like the transwell system won't work for you as you want to look at morphology and transwells aren't good for imaging.
Here's what I would recommend:
1) Separate microglia from astrocytes as normal
2) Spin down and seed on PDL coated 12 or 24-well plates (I would recommend a density between 0.5 and 0.7 million cells per mL when seeding). Culture overnight.
3) Harvest media conditioned for 24-72h by an astrocyte culture, spin at >10,000xg for 5 minutes to pellet any cell debris and retain the supernatant. You can probably sterile-filter this media through a 0.22um filter and store it for a week at 4 degrees before use.
4) Aspirate microglial media and replace with conditioned media.
5) Incubate - the timescale may need optimising. I would maybe start with a time course of between 4h and 24h
6) Perform assays. For morphology changes, you could simply live-image the cells. Alternatively you could culture them on coverslips, fix at the end of the treatment and stain with phalloidin or Iba1 etc...
