Infecting cells with C. trachomatis
You will need:
- McCoy cells grown until just confluent
- Cell culture medium (e.g. DMEM) with 10% FCS
- Cell culture flasks/trays
- Cycloheximide
- Gentamicin
- Tissue culture flasks/trays
- Centrifuge (capable of 754 x g)
- Class I or II safety cabinet
- Incubator at 37oC with 5% CO2
Protocol
- Take DMEM out of fridge and allow to reach room temperature; add cyclohexamide to desired volume of medium at 1µg/ml final concentration, and gentamicin at 10µg/ml
- Take flask of just confluent cells (~90%) from CO2 incubator
- Wipe surfaces, gloves and necks of all bottles with 70% IMS
- Pour medium off of cells
- Add inoculum to 5ml of medium (if using 25cm2 flask), mix gently and add to cell monolayer
- For LGV strains, loosen lids and place in 37oC / 5% CO2 incubator for 48 hours
- For other strains, centrifuge for 30 minutes at 754 x g before incubating to enable infection of cells