The overall goal of the protocol is to isolate primary microglia cells from live adult human brain tissues. In our case, this was collected as surgical window during surgery. This is accomplished by initially collecting the brain tissues in ice cold PBS, the tissue is then diced into 1-mm cube small pieces, and I just did with the help of trypsin EDTA.
Following this, the cells are harvested by centrifugation and plated in a flask suitable for androgen cell culture for 48 hours. Cells are harvested once more from the flask and cultured till further use. Primary human microglia cells can now be characterized by using immunocytochemistry and used for further experiment.
The main advantage of our protocol for isolating microglia cells from adult human brain tissues is that it effectively provides primary adult human microglia cells in a very cost-effective way. The protocol is robust and it reduces the loss of cells by reducing the number of steps being used in existing protocols. Collect the tissue in a 50-mL Falcon tube containing 10 mL of ice cold aCSF.
Wipe the collection tube carefully with 70%alcohol and transfer to an aseptic laminar air flow chamber. Discard the aCSF carefully and weigh tissue in the Falcon tube. Calculate the approx weight of tissue by deducing the weight of empty Falcon tube.
Warm fresh aCSF to 37 degrees centigrade. Keep the tissue in warm aCSF for five minutes. This step is critical to avoid cell death.
Discard aCSF carefully. And wash tissue once with 1X PBS warmed to 37 degrees centigrade. Ensure all blood is washed away with repeated PBS washes as needed.
Incubate the tissue in warm PBS for 5 minutes. Discard half of the PBS carefully. Transfer tissue along with remaining PBS to a sterilized Petri dish.
Remove remaining PBS carefully with 1-mL pipette. This will prevent loss of tissue. Dice that issue into at least 1-mm cube small pieces using a sterile scalpel.
Add 2 mL of 0.25%trypsin EDTA to Petri dish and wash the plate thoroughly with the help of pipette. Transfer the diced tissue to a 50-mL Falcon tube containing 10 mL per gram tissue of 0.25%trypsin EDTA. Mix by pipetting through a 10-mL serological pipette.
Incubate the tube on a shaker for 30 minutes at 37 degrees centigrade and the speed of 250 rpm. Add 10 mL of neutralizing medium to neutralize trypsin. Mix thoroughly with a 10-mL serological pipette.
Centrifuge the tube at 2000G at 4 degrees centigrade for 10 minutes. Discard the supernatant and resuspend the pellet in 1 mL of culture medium. Lay the cells in a T25 flask suitable for androgen cells and add 4 mL of additional culture medium.
Culture medium will contain 20%L929 supernatant and 1%streptomycin. It is recommended to add L929 supernatant separately in flasks instead of adding to the stock culture medium. We are finished with the flask to homogeneously dispose the tissue.
Avoid bringing the media to the neck of the flask while shaking as this may increase the chances of contamination. Incubate the flask at 37 degrees centigrade with 5%CO2 for 48 hours. Collect the media from T25 culture flask prepared on day 0 in centrifuge tubes.
Centrifuge the collected media at 1, 466G at 4 degrees centigrade for 4 minutes. While the collected media is being centrifuged, wash the day 0 flask once with 1X PBS. Shake the flask gently to remove any remnant tissue fragments left.
Avoid harsh shaking of the flask as any remnant fragments will not adversely affect the culture. Add 5 mL of fresh culture media to the flask. Discard the supernatant from centrifuged tubes.
Add 1 mL of culture medium to one of the tubes and mix thoroughly with pipette. Serially add the mixed media with cells to other tubes and mix thoroughly. Lay the cells in a separate T25 flask suitable for androgen cells.
Add 4 mL of fresh culture media to the flask. Incubate both the flasks at 37 degrees centigrade with 5%CO2 for 48 hours. Discard the media from both the flasks and add fresh 5 mL culture media.
Incubate the flasks at 37 degrees centigrade at 5%CO2 for 48 hours. On sixth day, cells will be ready for further experiments. In the images represented here.
First panel of the section A astrocytes stained with GFAP, green in color. In the second panel, of section A microglia stain with RCA, green in color. In the section B microglia stained with RCA, which is green in color, and astrocytes are stained with GFAP, red in color.
Overlay of the images shows microglia and astrocytes together. It is clear from the images that majority of the cells in the culture prepared are microglia. The images were quantified by blinded control and result is represented in section C.The results showed that about 80%of the cells in the culture prepared are microglia cells.
This technique can be performed in about one and a half hour. Following this procedure should have a good understanding of how do I select priming microglia from adult human brain tissues, which can be further used for downstream experiments.
