FBA-staining of bacteria

Materials

(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”