J774A.1 and HMC3 Cell Culture

Author

Brian

The following protocol is designed to grow and maintain mammalian cell culture colony.

Reagents

  1. J774A.1 (TIB-67) - Peripheral Mouse Macrophage cell line

  2. HMC3 (CRL-3304) - Human Microglia cell line

  3. RPMI and DMEM

    1. With Glutamine, pyruvate, glucose and sodium bicarbonate

    2. Can be purchased through Aggiebuy or UC Davis Scientific store

  4. Fetal Bovine Serum (FBS)

    1. US or USDA acceptable regions

    2. Can be purchased through Aggiebuy

  5. Penicillin-Streptomycin (10,000 U/mL) (ThermoFisher, 15140122)

    1. Can be purchased through Aggiebuy

  6. Trypsin-EDTA (0.05%), phenol red

    1. To dissociate cells for passing

    2. Can be purchased through Aggiebuy

  7. T75 flasks for adherent culture

    1. Any flasks that works for adherent cells

    2. For example Corning

  8. Glass Pasteur Pipets - Vacuum old media

  9. 10 mL, 25 mL glass pipette - Dispense media

    1. Can be purchased on AggieBuy or UC Davis Scientific Store

  10. Cell scraper (J774A.1)

    1. Can be purchased through AggieBuy

Complete Media

  1. For DMEM or RPMI

    1. 500 mL of Media (DMEM or RPMI)

    2. 50 mL of FBS (10%)

    3. 5 mL of Antibiotics (1%)

  2. J774A.1 can be grown in complete RPMI or DMEM

  3. HMC3 can only be grown in complete DMEM

Method - J774A.1 or HMC3

Check liquid nitrogen tank (Rack 3, last box at the bottom of the rack) for more any ampoules.

  1. Thaw cells for ~5min in 37C

    1. There should be at least 1mL of cells

  2. Re-suspend cells in 9 mL of complete media

    1. Total volume ~10 mL

  3. Spin at 300g for 5min

  4. Vacuum old media using glass pipette

  5. Re-supend cells to 20 mL then transfer to T75 flask (or 40 mL using T175 flasks)

Checking Cell Confluence

  1. Cells should be passaged once cells reaches 70-80% confluency.

HMC3 Low and High Density

J774A.1 Low and High Density

Passaging Cells and changing Media

  1. Media should be changes every 2 days. Cells that are not confluent by day 2 (<70%) can have their old media replaced with new complete media, then check the next day to see if cells reaches confluence.

To passage cells (HMC3)

  1. Remove old media

  2. Dispense 5 mL of Trypsin-EDTA

  3. Incubate in 37C incubator for 5-10 min

  4. Dispense 5 mL of complete media into flask

    1. Total volume is 10 mL (5 mL from trypsin-EDTA and 5 mL from complete media)

  5. Transfer 10 mL of cell solution from flask to a 50 mL conical tube

  6. Spin at 300g for 5min

1:4 or 1:5 split

  1. Example (1:4 split):

    1. Resupend cell pellet with 20 mL of complete media then transfer 5 mL of cell supsension into 15 mL of complete media. Mix with pipette then transfer to a T75 flasks.

    2. For a 1:4 split, you can passage 1 confluent flask to create up to 4 new flasks.

      1. It’s ok to throw away any extra cells you don’t need it, but keep the culture flowing.

To passage cells (J774)

  1. Remove ~10mL of media

  2. Scrape cells with cell scraper

  3. Transfer cells to 50 mL conical tube

  4. Spin 300g for 5min

  5. Resupsend cells in 20mL of complete media

  6. Split 1:4 or 1:5

Good Practice

  1. Keep passage low <10 passages when executing cell-based assays, but for learning, you can keep the culture going passed passage 10.

  2. Passage cells into new flasks, or wash old flasks with PBS.

  3. Always spray down hood and equipment with CaviCide or 70% ethanol.

  4. Keep water bath clean, replace old water with milliQ water

  5. If cells looks funny (won’t adhere, cell death, weird shape), throw away and thaw new cells.