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Catalog Number PHM001Product Name Primary Human BrainMicroglia CellsStorage Liquid NitrogenProduct Format Frozen vialCells Number >90% confluent in Frozen Vial500.000 cells*Caution: The handling of human derived products has the potential to be biologically hazardous. All Cell strains tested negative for HIV, HBV,and HCV DNA in diagnostic tests. Proper precautions must be taken to avoid exposure. Always wear proper protective equipment (Gloves,safety glasses, etc.) when handling these materials. We recommend following the universal procedures for handling products of human originas the minimum precaution against contamination.GENERAL INFORMATION:Microglia, one of the glial cell types in the CNS, is an important integral component ofneuroglial cell network. They have been observed in the brain parenchyma from the earlystage of development to the mature state. Microglia act as brain macrophages whenprogrammed cell death occurs during brain development or when the CNS is injured orpathologically damaged.Microglia can be considered as the main cell in brain immune surveillance, can presentantigens in the molecular context of MHC class II expression to CD-4 positive T cells, arecapable of Fc mediated phagocytosis, and share many common antigens with hemopoieticand tissue macrophages. Furthermore, there is accumulating evidence that microglia areinvolved in a variety of physiological and pathological processes in the brain by interactingwith neurons and other glial cells and through production of biologically active substancessuch as growth factors, cytokines, and other factors. Human brain microglia cells areisolated from healthy human brain tissue. After purification, PHM001 are cryopreservedand delivered frozen. PHM001 are ready to plate in a culture vessel for experiment, but notrecommended for expanding or long term cultures since the cells do not proliferate inculture. It is recommended to use Alpha-Glia Expansion Medium for the culturing ofPHM001
Characterization of the cells:Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested,fixed then analyzed by flow cytometry using anti-CD68 (ED-1) antibodies. Labeled cells arerepresented by the black shaded populations, whereas the unlabeled cells are depicted by thegrey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).Cytoplasmic F4/80: >98% positive by immunofluorescenceCD68: >98% positive by immunofluorescenceThe cells maintained microglial specific markers such as NGF, CD68 and TREM2 asdemonstrated by RT-PCR. These cells are suitable for studies of human microglia inhealth and disease.
Handling of Arriving Cells:When you receive the cells in a frozen vial, you can transfer the vial of cells into a -80ºCfreezer for short period storage or a liquid nitrogen tank for long term storage. Thawthe cells in a 37°C water bath, and then transfer the cells into a T25 flask pre-coatedwith poly-L-lysine as described in details in Subculture Protocol.Subculture Protocol:1. Prepare a poly-L-lysine coated flask (2μg/cm2 , T-25 flask is recommended). Add 5ml of sterile water to a T-25 flask and then add 9μl of poly-L-lysine stock solution10mg/ml. Leave the flask in incubator overnight (minimum one hour at37°Cincubator).2. Rinse the poly-L-lysine coated flask with sterile water twice and add 7 ml ofcomplete 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 contentsare completely thawed. Remove the vial from the water bath immediately, wipe itdry, rinse the vial with 70% ethanol and transfer it to a sterile field. Remove thecap, being careful not to touch the interior threads with fingers. Using 1 mlEppendorf pipette gently resuspend the contents of the vial.
5. Dispense the contents of the vial into the equilibrated, poly-L-lysine coated culturevessels. A seeding density of ≥10,000 cells/cm2 is recommended. Note: Dilution andcentrifugation of cells after thawing are not recommended since these actions aremore harmful to the cells than the effect of DMSO residue in the culture. It is alsoimportant that cells are plated in poly-L-lysine coated culture vessels that promote cellattachment.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 hasbeen initiated. Change the growth medium the next day to remove the residual DMSOand unattached cells, then every other day thereafter.Maintenance of Culture:1. Change the medium to fresh supplemented medium the next morning afterestablishing a culture from cryopreserved cells.2. Change the medium every two to three days thereafter.