View source for OPTIC (Opa protein triggered integrin clustering)

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