OPTIC (Opa protein triggered integrin clustering) - Revision history

ESO wikiadmin at 08:47, 20 May 2025

2025-05-20T08:47:45Z

← Older revision		Revision as of 10:47, 20 May 2025
Line 20:		Line 20:
	==== '''Day1:''' ====		==== '''Day1:''' ====

	~~====~~ Seed HEK293T cells 1:40 in 6 well plate. 1 well for each transfection approach ~~====~~		* Seed HEK293T cells 1:40 in 6 well plate. 1 well for each transfection approach

	==== '''Day2:''' ====		==== '''Day2:''' ====

ESO wikiadmin at 08:47, 20 May 2025

2025-05-20T08:47:26Z

← Older revision		Revision as of 10:47, 20 May 2025
Line 36:		Line 36:

	==== '''Day4:''' ====		==== '''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		* 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 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
	§ 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		* Wash 3x with PBS
			* Add 300 µl of blocking solution, incubate for 10 min.
	§ Add 300 µl of permeabilization solution and incubate for 6 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		* Wash 3x with PBS
			* Add 300 µl of blocking solution and incubate 10 min
	§ Add 300 µl of blocking solution, incubate for 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
	§ 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)		* Wash 3x with PBS
			* Transfer coverslips to microscope slide (mounting medium from Dako)
	§ Incubate for 1h in humidified chamber		* Analyze at confocal microscope

	§ 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

ESO wikiadmin: Created page with "{| class="wikitable" |+ !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..."

2025-05-20T08:43:59Z

Created page with "{| class="wikitable" |+ !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..."

New page

{| class="wikitable"
|+
!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<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