ESO wikiadmin: Created page with "{{Protokoll-Mix|Inhalt=(5-(6)-carboxyfluorescein succinimidyl ester (CFSE, FAM SE, Fluorescein)
NHS-Biotin
PBS++ (PBS, 45 nM CaCl 2, 35 nM MgCl 2)
4% Paraformaldehyde (PFA)
Blocking solution (10% FCS in PBS ++)
PBS
Streptavidin-AlexaFluor647}} == Procedure == * Dilute 0.2 mg NHS-Biotin in 1 ml PBS (prewarm Biotin to RT before opening the tube) * Collect bacteria from plate in PBS, centrifuge for 4 min, 4000 rpm, and resuspend the pellet in 700 l PB..."

2025-06-02T08:28:41Z

Created page with "{{Protokoll-Mix|Inhalt=(5-(6)-carboxyfluorescein succinimidyl ester (CFSE, FAM SE, Fluorescein) NHS-Biotin PBS++ (PBS, 45 nM CaCl 2, 35 nM MgCl 2) 4% Paraformaldehyde (PFA) Blocking solution (10% FCS in PBS ++) PBS Streptavidin-AlexaFluor647}} == Procedure == * Dilute 0.2 mg NHS-Biotin in 1 ml PBS (prewarm Biotin to RT before opening the tube) * Collect bacteria from plate in PBS, centrifuge for 4 min, 4000 rpm, and resuspend the pellet in 700 l PB..."

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{{Protokoll-Mix|Inhalt=(5-(6)-carboxyfluorescein succinimidyl ester (CFSE, FAM SE, Fluorescein) NHS-Biotin PBS++ (PBS, 45 nM CaCl 2, 35 nM MgCl 2) 4% Paraformaldehyde (PFA) Blocking solution (10% FCS in PBS ++) PBS Streptavidin-AlexaFluor647}}

== Procedure ==

* Dilute 0.2 mg NHS-Biotin in 1 ml PBS (prewarm Biotin to RT before opening the tube)
* Collect bacteria from plate in PBS, centrifuge for 4 min, 4000 rpm, and resuspend the pellet in 700 l PBS
* Mix with 700 l Biotin solution
* Add CFSE at a final concentration of 0.5 g/ml
* Incubate for 20 min at RT with shaking (protected from light)
* Wash 3 times with 1 ml PBS (4 min, 4000 rpm)
* Infect the cells with desired MOI and incubate for desired time (standard: 1h)
* Aspirate medium and wash once with 350 ml PBS ++. Never aspirate medium completely, keep the cells always wet. Add PBS++ carefully over the edge of the well to avoid detachment of the cells!
* Add 500 l PFA, incubate 20 min at RT in the dark (fixation)
* Wash 3 times with PBS ++
* Block with 300 l blocking solution for 10 min at RT (dark)
* Add 25 l of Streptavidin-AlexaFluor647 (if using Cy5-tagged antibody: 1:100 – 1:400 in blocking solution)
* Incubate 1h in a wet chamber at RT (dark)

For further proceeding: go to “Immunofluorescence staining”

FBA-staining of bacteria - Revision history