Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

This protocol describes a method that is focused on extracting in parallel the two most abundant glial cell populations, astrocytes and microglia, from postnatal mice. Our method differs from others previously described protocols for astrocyte and microglia isolation because we focus on obtaining and isolating both cell types in the same extraction at the same condition, thus optimizing the use of experimental animals and improving the study of relative roles for these cells in immunological contexts. This protocol can be useful for better investigating microglia and astrocyte roles in many contexts and diseases in the central nervous system such as Alzheimer, Parkinson, and infections by for example protozoa parasites or viruses.

Obtaining and isolating both cell types from the same extraction is desirable to evaluate the comparative role of each cell subset immunological contexts such as infection or inflammation. People should be careful especially during brain removal and nervous tissue washes, but I believe that with this protocol and the video shot, people will not struggle. This protocol can be long depending on the number of animals to extract the cortices so be prepared to spend about four hours or more.

On day one, prepare pure HBSS and HBSS plus 10%heat-inactivated FBS. Prepare supplemented DMEM F12 and filter it with a 0.22 micron filter. Sterilize all surgical instruments in an autoclave and use 70%ethanol during the procedure.

Put newborn mice in a sealed chamber containing cotton soaked with isoflurane for five minutes for profound anesthesia. Spray the mouse pup with 70%ethanol and decapitate the animal with scissors. Make a sagittal cut with scissors along the cranium to open it and expose the brain.

Remove the brain using a micro spatula to maintain the brain integrity. Place the brain in a dry six centimeter diameter Petri dish. Using a micro spatula, remove the olfactory bulb and cerebellum.

Move the cortex to another Petri dish containing two milliliters of HBSS plus 10%FBS. Cut the brain tissue into small pieces with sterile scissors and using a P1000 micropipette, transfer each brain tissue with HBSS plus 10%FBS to different 15 milliliter conical tubes. If necessary, use HBSS plus 10%FBS to fill the final volume to two milliliters.

Wash the cut brain tissue with three milliliters of HBSS plus 10%FBS for each tube. After decantation, carefully remove the supernatant. Repeat the wash with HBSS plus 10%FBS three additional times.

Then wash with three milliliters of pure HBSS three times. After decantation, carefully remove the supernatant. Next, add three milliliters of trypsin and place the tube in a water bath at 37 degrees Celsius for 30 minutes gently shaking the tubes every five minutes.

This digests the collected tissue. Avoid bubbles. To inactivate the trypsin, wash the tissue with three milliliters of HBSS plus 10%FBS and after decantation of the tissue, carefully remove the supernatant.

Repeat this wash three times. Then wash the tissue with three milliliters of pure HBSS and repeat two additional times. After decantation of the tissue, carefully remove the supernatant.

Add seven milliliters of pure HBSS and homogenize the tissue through successive passages in pipettes, first with a 10 milliliter serological pipette, followed by a five milliliter pipette, and finally with a P1000 micropipette. After homogenization, centrifuge the tubes at 450 times g for five minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet with four milliliters of HBSS plus 10%FBS for each tube.

Transfer the cells to a commercially pretreated T75 flask for optimal cell culture adhesion. Place the flask in an incubator at 37 degrees Celsius at 5%carbon dioxide for 30 minutes for adherence. Then add 10 milliliters of supplemented DMEM F12 to the flask and incubate at 37 degrees Celsius and 5%carbon dioxide for two nights.

On day three, remove seven milliliters of culture medium from the T75 flask and add seven milliliters of fresh supplemented DMEM F12. Return the flask to the incubator. On day five, to remove debris and nonadherent cells, exchange all medium from the T75 flask with 14 milliliters of fresh supplemented DMEM F12.

Then every 48 hours, remove six milliliters of the supernatant and add seven milliliters of fresh supplemented DMEM F12 medium until day 14 of culture. On day 14, after removing six milliliters of supernatant and adding seven milliliters of fresh supplemented DMEM F12 medium, close the T75 flasks tightly with plastic wrapping. Place them in the floor orbital shaker at 200 RPM and 37 degrees Celsius overnight to mechanically dissociate microglia from astrocytes.

On day 15, take the flasks from the shaker and vigorously wash them with their own medium in order to optimize cell dissociation and harvest the maximum number of microglia. Then collect the supernatant and transfer it to a 50 milliliter conical tube. Next, add four milliliters of trypsin to each flask and incubate for five minutes at 37 degrees Celsius in order to detach the astrocytes.

Then add five milliliters of supplemented DMEM F12 to inactivate the trypsin. Wash the flasks with their own medium and transfer the contents to a 50 milliliter conical tube. Centrifuge all tubes containing dissociated microglia or astrocytes at 450 times g for five minutes at four degrees Celsius.

Discard the supernatant. Resuspend the microglia pellet in one milliliter of supplemented DMEM F12 and the astrocytes in 10 milliliters of supplemented DMEM F12. Proceed to cell counting in a Neubauer chamber or an automatic cell counter.

Plate the cells with supplemented DMEM F12 at the desired density in a pretreated flat bottom cell culture plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours to allow them to attach. On the 14th day, glial cell culture underwent mechanical dissociation.

It was observed with a purity of 89.5%for the astrocyte population and 96.6%for the microglia population. Astrocytes and microglia from C57BL6 mice were infected with T.cruzi Y.After two hours, 48 hours, and 96 hours, chambers were fixed with methanol and stained with cell nuclease marker DAPI and anti-GFAP. The use of genetically modified parasites expressing fluorescent reporters or parasites labeled with specific fluorescent antibodies improved the immunofluorescence microscopy since they are better distinguished from the cell nucleus.

Two examples are presented here. T.gondii RH strain constitutively expressing YFP and T.cruzi Y strain stained with non-commercial monoclonal antibody 2C2 anti-SSP4 protein. It is important to carefully wash the cells to not lose any cell aggregates.

After glial cells infection, other parameters can be evaluated such as ELISA for cytokine production present in the supernatant, Western blotting for protein expression, and real time quantitative PCR for RNA transcription evaluation. Many questions related to the function of glial cells, for example their metabolism, immune response, and cell biology itself can be addressed and benefited by this technique. All protozoa parasites are able to infect human cells as well as culture mouse cells so it is important to be careful when handling this parasite.