OPTIC (Opa protein triggered integrin clustering)

Materials
1x PBS
Trypsin/EDTA
GC-Agar plates with CAM/ERM and without antibiotics Coverslips (round, 12 mm)
24 well plate
HEK culture medium (DMEM + 10% CS) Suspension medium (DMEM + 0.25% BSA) 4% paraformaldehyde (PFA) in PBS
Permeabilization solution (0.4% Triton-X-100 in PBS) Blocking solution (10% heat inactivated CS in PBS) Mounting medium

Procedure

Day1:

• Seed HEK293T cells 1:40 in 6 well plate. 1 well for each transfection approach

Day2:

• Prepare transfection approach according to transfection protocol for HEK cells described previously.
• Use 2.5 µg of the OPTIC receptor construct or the control plasmid + 2.5 µg of the cytoplasmic protein of interest.
• Add 2 µl of chloroquine to the cells and transfect cells with 200 µl of the transfection approach.
• Exchange medium after 6-8h
• In the evening streak out N. gonorrhoeae (N309) on GC agar plates containing Chloramphenicol and Erythromycin using a three eyelet smear

Day3:

• Coat coverslips in 24 well plate with poly L-Lysine (10 µg/ml) and incubate over night at 4°C
• In the afternoon, not more than 24 h after plating the bacteria, select a number of opaque colonies and streak them further on GC agar w/o antibiotics

Day4:

• In the morning seed 2*10^5 HEK cells on coated coverslips in suspension medium and let adhere for 2h.
• In the meantime resuspend bacteria from plate in 1ml 1xPBS and stain with pacific blue (1:1000, 30 min, 37°C). Wash 3x with PBS and determine cell number using the formula: OD550 x 8.6 x 10^7 bacteria/ml
• After 2h infect HEK cells with MOI 20-50 in suspension medium for 1h at 37°C
• After infection remove medium and fix cells with 4% PFA/PBS
• Wash 3x with PBS
• Add 300 µl of permeabilization solution and incubate for 6 min
• Wash 3x with PBS
• Add 300 µl of blocking solution, incubate for 10 min.
• Add 25 µl of 1^st antibody at appropriate dilution in blocking solution. Apply to the center of the coverslip (make sure coverslip does not touch wall of the well)
• Incubate for 1h in humidified chamber
• Wash 3x with PBS
• Add 300 µl of blocking solution and incubate 10 min
• Add 25µl of 2^nd antibody at appropriate dilution in blocking solution in the center of the coverslip (make sure that coverslips does not touch wall of the well)
• Incubate for 45 min in humidified chamber
• Wash 3x with PBS
• Transfer coverslips to microscope slide (mounting medium from Dako)
• Analyze at confocal microscope