Immortalized Human Brain Microglia Cells

Catalog Number HBMCs001
Product Name Immortalized Human Brain
Microglia Cells
Storage Liquid Nitrogen
Product Format Frozen vial
Cells Number >90% confluent in Frozen Vial
1,000,000 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 origin
as the minimum precaution against contamination.
GENERAL INFORMATION:
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, HBMCs001 are cryopreserved
and delivered frozen. HBMCs001 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 Microglia Growth Medium for the culturing of HBMCs.

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 are
represented by the black shaded populations, whereas the unlabeled cells are depicted by the
grey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).
Cytoplasmic F4/80: >98% positive by immunofluorescence
CD68: >98% positive by immunofluorescence
The cells maintained microglial specific markers such as NGF, CD68 and TREM2 as
demonstrated by RT-PCR. These cells are suitable for studies of human microglia in
health and disease.

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.
Subculture Protocol:
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 Microglia Growth Media 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.
Maintenance of Culture:
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.