labelling of Chlamydia bacteria

Rafael Nunez rafaeln at vetvir.unizh.ch
Tue Sep 19 01:43:09 EST 2000


Hi Andreas, you may consider labeling with CFSE, here is our protocol

Protocol for Staining with CFSE.
1-	Cells in logarithmic growth phase are washed with PBS (x 3).
2-	Resuspend them in 2 ml of PBS without FCS or protein supplement.
Final concentration: 6-10 x 107 cells/ml.
3-	 Add 2.8 mg/ml of CFSE.
4-	Incubated cells at 37°C for 10 minutes with mixing 3 to 4 times.
5-	Add several volumes of ice-cold medium to stop CFSE binding.
6-	 centrifugation for 10 minutes (4°C)
7-	Resuspend pellet cells with fresh medium.
8-	Adjust cells density to your desidered concentration
9-	Optional: Add medicament(s) at variable concentrations (0 through
800 mg/ml).
10-	Determine the CFSE fluorescence by flow cytometry immediately after
staining, and after 24, 48, 72, and 96 hours.
I hope it will help
Regards Rafael

>Hi flowers,
>
>does anyone have experience in permanent fluorescent labelling of live
>bacteria, in particular Chlamydia trachomatis ? We would like to quantify
>infection rates in monocytes.
>
>Any help would greatly be appreciated.
>
>Thanks in advance!
>
>
>Andreas Thiel
>
>
>Dr. Andreas Thiel
>Clinical Immunology
>Schumannstr. 21/22
>10117 Berlin
>Germany
>
>phone	  ++49 30 28460 682
>
>
>email:thiel at drfz.de


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    ___/\_    | Rafael Nunez		      mailto:rafaeln at vetvir.unizh.ch
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