HAPI大鼠小胶质细胞cell line细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心

HAPI大鼠小胶质细胞cell line细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心
Description HAPI Rat Microglial Cell Line
Alternate Names • HAPI (Highly Aggressively Proliferating Immortalized) cell line
Background Information Microglia are the primary immune cells of the central nervous system (CNS), and accounts for 10-15% of all cells found within the brain. Microglial play an important role in maintaining the health of the CNS by the removal of pathogens, infectious agents and damaged cells through phagocytosis. Microglial have also been extensively studied for their harmful roles in a variety of neurodegenerative disease including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis.

The HAPI Rat Microglial Cell Line is a spontaneously immortalized cell line derived from primary microglia-enriched cultures prepared from 3-day-old rat brains (1). HAPI (highly aggressively proliferating immortalized) cells retain characteristics of microglia including marker expression, proinflammatory cytokine secretion in response to LPS, and phagocytic activity (1). These cells provide an alternative model to primary microglial cells.

Reference:
1. Cheepsunthorn, P., Radov, L., Menzies, S., Reid, J., & Connor, J. R. (2001). Characterization of a novel brain‐derived microglial cell line isolated from neonatal rat brain. Glia, 35(1), 53-62.
Applications
Application HAPI rat microglial cells are highly proliferating and retain the phenotypic characteristics of microglia.
Key Applications • Cell Culture
• Cell Based Assays

Low Glucose DMEM (Sigma Cat. No. D6046), 5% FBS (EMD Millipore Cat. No. ES-009-B) and 1X Penicillin-Streptomycin Solution (EMD Millipore Cat. No. TMS-AB2-C).


Protocols
Thawing Cells
1. Do not thaw the cells until the recommended medium is on hand. Cells can grow on normal tissue culture ware surfaces without any additional coating.

Cells are thawed and expanded in Low Glucose DMEM (Sigma Cat. No. D6046), 5% FBS (EMD Millipore Cat. No. ES-009-B) and 1X Penicillin-Streptomycin Solution (EMD Millipore Cat. No. TMS-AB2-C).
2. Remove the vial of frozen HAPI cells from liquid nitrogen and incubate in a 37°C water bath. Closely monitor until the cells are completely thawed. Maximum cell viability is dependent on the rapid and complete thawing of frozen cells.
IMPORTANT: Do not vortex the cells.
3. As soon as the cells are completely thawed, disinfect the outside of the vial with 70% ethanol. Proceed immediately to the next step.
4. In a laminar flow hood, use a 1 or 2 mL pipette to transfer the cells to a sterile 15 mL conical tube. Be careful not to introduce any bubbles during the transfer process.
5. Using a 10 mL pipette, slowly add dropwise 9 mL of HAPI Expansion Medium (Step 1 above) to the 15 mL conical tube.
IMPORTANT: Do not add the entire volume of media all at once to the cells. This may result in decreased cell viability due to osmotic shock.
6. Gently mix the cell suspension by slowly pipetting up and down twice. Be careful not to introduce any bubbles.
IMPORTANT: Do not vortex the cells.
7. Centrifuge the tube at 300 x g for 2-3 minutes to pellet the cells.
8. Decant as much of the supernatant as possible. Steps 5-8 are necessary to remove residual cryopreservative (DMSO).
9. Resuspend the cells in 10-15 mL of HAPI Expansion Medium.
10. Transfer the cell mixture to a T75 tissue culture flask.
11. Incubate the cells at 37°C in a humidified incubator with 5% CO2.
12. The next day, exchange the medium with 10-15 mL of fresh HAPI Expansion Medium. Exchange with fresh medium every two to three days thereafter.
13. When the cells are approximately 90-95% confluent, they can be dissociated with Accutase (EMD Millipore Cat. No. SCR005) or trypsin-EDTA (EMD Millipore Cat. No. SM-2003-C) and further passaged or, alternatively, frozen for later use.
.
Subculturing Cells
Note: Upon first thawing from liquid nitrogen storage, HAPI cells may grow slowly at first. Once established, HAPI cells will proliferate rapidly. Cells will appear mostly small and round with some having the traditional appearance of microglia, i.e. more flattened with processes. These cells do not have contact inhibition so they may grow on top of each other if allowed. There are a certain percentage of floaters, which is not dependent upon the confluence of the cells. Established HAPI cultures comprise a combination of flat process-bearing adherent cells and loosely attached clusters of round cells without processes.
1. Carefully remove the medium from the T75 tissue culture flask containing the confluent layer of HAPI cells.
2. Rinse the T75 flask twice with 10 mL 1X PBS. Aspirate after each rinse.

3. Apply 3-5 mL of Accutase or trypsin-EDTA solution and incubate in a 37°C incubator for 3-5 minutes.
4. Inspect the flask and ensure the complete detachment of cells by gently tapping the side of the flask with the palm of your hand.
5. Add 8 mL of HAPI Expansion Medium to the plate.
6. Gently rotate the flask to mix the cell suspension. Transfer the dissociated cells to a 15 mL conical tube.
7. Centrifuge the tube at 300 x g for 3-5 minutes to pellet the cells.
8. Discard the supernatant, then loosen the cell pellet by tapping the tip of the tube with a finger.
9. Apply 2 mL of HAPI Expansion Medium to the conical tube and resuspend the cells thoroughly.
IMPORTANT: Do not vortex the cells.
10. Count the number of cells using a hemocytometer.
11. Plate the cells to the desired density (typical split ratio is 1:7 – 1:9).
Cryopreservation of Cells
HAPI Rat Microglial Cell Line may be frozen in the expansion medium plus 10% DMSO using a Nalgene slow freeze Mr. Frosty container.
【Supplier来源】BioVector NTCC Inc.
TEL：+86-010-53513060
【Website网址】 http://www.biovector.net