Microglia, one of the glial cell types in the CNS, is an important integral component of neuroglial cell network. They have been observed in the brain parenchyma from the early stage of development to the mature state. Microglia act as brain macrophages when programmed cell death occurs during brain development or when the CNS is injured or pathologically damaged. Microglia can be considered as the main cell in brain immune surveillance, can present antigens in the molecular context of MHC class II expression to CD-‐4 positive T cells, are capable of Fc mediated phagocytosis, and share many common antigens with hemopoietic and tissue macrophages. Furthermore, there is accumulating evidence that microglia are involved in a variety of physiological and pathological processes in the brain by interacting with neurons and other glial cells and through production of biologically active substances such as growth factors, cytokines, and other factors. Human brain microglia cells are isolated from healthy human brain tissue. After purification, PHM001 are cryopreserved and delivered frozen. PHM001 are ready to plate in a culture vessel for experiment, but not recommended for expanding or long term cultures since the cells do not proliferate in culture. It is recommended to use Alpha-‐Glia Expansion Medium for the culturing of PHM001.
Handling Of Arriving Cells
When you receive the cells in a frozen vial, you can transfer the vial of cells into a -‐80ºC freezer for short period storage or a liquid nitrogen tank for long term storage. Thaw the cells in a 37°C water bath, and then transfer the cells into a T25 flask pre-‐coated with poly-‐L-‐lysine as described in details in Subculture Protocol.
1. Change the medium to fresh supplemented medium the next morning after establishing a culture from cryopreserved cells. 2. Change the medium every two to three days thereafter.
1. Prepare a poly-‐L-‐lysine coated flask (2μg/cm2 , T-‐25 flask is recommended). Add 5 ml of sterile water to a T-‐25 flask and then add 9μl of poly-‐L-‐lysine stock solution 10mg/ml. Leave the flask in incubator overnight (minimum one hour at37°C incubator). 2. Rinse the poly-‐L-‐lysine coated flask with sterile water twice and add 7 ml of complete medium to the flask. Leave the flask in the hood and go to thaw the cells. 3. Warm Alpha Glia Expansion Medium before thawing the cells. 4. Place the vial in a 37°C water bath, hold and rotate the vial gently until the contents are completely thawed. Remove the vial from the water bath immediately, wipe it dry, rinse the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful not to touch the interior threads with fingers. Using 1 ml Eppendorf pipette gently resuspend the contents of the vial.5. Dispense the contents of the vial into the equilibrated, poly-‐L-‐lysine coated culture vessels. A seeding density of ≥10,000 cells/cm2 is recommended. Note: Dilution and centrifugation of cells after thawing are not recommended since these actions are more harmful to the cells than the effect of DMSO residue in the culture. It is also important that cells are plated in poly-‐L-‐lysine coated culture vessels that promote cell attachment. 6. Replace the cap or cover, and gently rock the vessel to distribute the cells evenly. Loosen cap if necessary to permit gas exchange. 7. Return the culture vessels to the incubator. 8. For best result, do not disturb the culture for at least 16 hours after the culture has been initiated. Change the growth medium the next day to remove the residual DMSO and unattached cells, then every other day thereafter.