Harvesting C. trachomatis
You will need:
- Cell scrapers
- 4SP buffer (for 250ml: Sucrose 34.23g in150ml sterile water; Na2HPO4 0.567g in 50ml sterile water; adjust pH to 7.1 and volume to 250ml. Filter sterilize. Store at 4oC)
- Cold 10% PBS
- Sterile glass beads
- Bijoux tubes
- Vortex
- Centrifuge (capable of 2850 x g)
- Class I or II safety cabinet
Protocol
- Take flask/tray with infected cell monolayer
- Scrape all cells up into the medium using a cell scraper (or a 1ml tip for wells)
- Transfer to a 50ml centrifuge tube. Centrifuge at 2850 x g for 10 minutes to concentrate the harvest (if from a flask. For small volumes from wells this step is not necessary)
- Remove supernatant and re-suspend pellet in 2ml of cold 10% PBS
- Transfer to a bijoux tube containing ~5 glass beads
- Vortex at high speed for 1 minute
- Transfer by Pasteur pipette to a 50ml centrifuge tube
- Centrifuge at 230 x g for 5 minutes to remove cell debris
- Transfer supernatant to a clean cryovial for storage; add an equal volume of 4SP buffer
- Store at -70 to -80oC