X-gal titration
You will need:
- X-gal staining solution
- 0.016g K3Fe(CN)6 (Potassium Ferricyanide)
- 0.021g K4Fe(CN)6 (Potassium Ferrocyanide)
- 0.004g MgCl2.6H2O (Magnesium Chloride 6-hydrate)
- 9.8ml sterile PBS
- Filter sterilize with 0.2μm filter and store at 4oC
- 50mg/ml X-gal stock
- In a glass bijoux: 0.05g X-Gal + 1ml DMF (Dimethylformamide)
- Wrap in foil, and store at -20oC
Method
- Serially dilute chlamydia stock in DMEM medium, in a 96 well tray (e.g. neat, 10-1, 10-2, 10-3, etc)
- Centrifuge as necessary and incubate at 37oC/5% CO2 until inclusions are ready
- Replace medium with 100μl PBS to wash; repeat once
- Replace with 100μl ice cold methanol; incubate at -20oC for 20 minutes
- Wash with 100μl PBS; repeat once
- Dilute required primary antibody as required in PBS - we can provide monoclonal antibodies for the chlamydia genus (LPS), C. trachomats (MOMP) and C. pneumoniae (MOMP) - see here for details
- Incubate overnight at 4oC, or for 40 minutes at 37oC
- Make up conjugate antibody dilution in PBS (i.e. we use 1:1000 dilution of Anti-mouse IgG, Heavy and Light chain specific (goat) β-galactosidase conjugate)
- Wash wells 3 x 2 minutes in PBS
- Add 100μl of conjugate per well
- Incubate for 1 hour at 37oC
- Wash wells 3 x 2 minutes in PBS
- Make up X-gal staining solution and X-gal stock (as above)
- For each 0.5ml of X-gal staining solution, add 10μl of X-gal stock
- Add 100μl to each well
- Incubate at 37oC for 4 hours
- Observe under microscope and count stained inclusions (it may help to visualise inclusions by replacing the staining solution with PBS)
- If required, store in PBS at 4oC