CagA phosphorylation assay and its semiquantitative analysis

H. pylori has a amazing capacity to change during growth. In order to keep the experiments reproducible and comparable, use relative fresh bacteria for all infection experiments.

On Friday afternoon, take from frozen stocks the bacteria by scratching the surface of the ice with the inoculation loop. A piece of "ice" will adhere to the loop. Do not distribute the material on the plate. Just place it on the agar and let H. pylori grow on the small drop of defrosted media, undisturbed. Place the agar plates to grow in an incubator with microaerophil conditions (10% CO₂, 5% O₂ and 75% N₂) at 37°C for the weekend.

On Monday, passage the bacteria to a new GC Cholesterol plate

, and pass them onto a new plate on Tuesday for Wednesday's work, where bacteria are fit enough to be used for infection.

The objective is to have a cellular confluency in the 6-well of 80-90% at the moment of infection. For you to achieve this, you have to know your cells and their multiplication speed. For AGS cells the usual splitting for 48 hours with synchronization (see below) was 1:8 (from a 90% - 100% confluent culture)

Remove serum-containing media (RPMI + 10% Inactivated FCS, Complete Media (CM)) and change it for media without serum (RPMI without serum) around 16 hours before infection, in order to synchronize cells in G₀.

For 1/4 of plate grown bacteria, collect all bacteria using a sterile cotton swab and introduce it in a tube containing at least 500 µl of PBS. Using circular movements, remove the bacteria culture attached to the cotton fibers using the PBS solution. The objective is to have as much bacteria in the solution, and reduce the amount of liquid attached to the cotton swab before discarding it.

Make a 1:100 dilution of bacteria suspension in PBS (10µl bacterial suspension in 990µl PBS) and measure its optical density (OD) in a densitometer at 550 nm.

Multiply the dilution factor to obtain the OD₅₅₀ / ml of suspension.

Calculate the amount of bacteria in your suspension taking into consideration that and OD₅₅₀ of 0,1 is equivalent to aprox. 3x10⁷ CFU/ml.

Multiplicity of Infection (MOI) of 60 means that you will add 60 bacteria for each cell in the well. For general calculations, each well of a 6-well cell culture plate has approx. 1x10⁶ adherent cells at a confluency of 90% to 100%

Remove the serum-free media from the cells and add 1 ml of complete media (CM) per well around 30 min before infection

Infect cells and incubate at 5% CO₂ 37°C for 3 to 4 hours.

Stop the infection by placing the plate on ice. For the rest of the procedure, maintain the samples cold.

Collect the media for cytokine measurements. If no cytokine measurements are necessary, remove the media with help of a vacuum pump. Add 1 ml PBS* (PBS with protease and phosphatase inhibitors). Prevent the well’s surface from drying out (crystal formation damages the cells).

With a cell scrapper detach the cells from the bottom of the plate.

Collect the cells suspension in 1,5 ml tubes. Centrifuge the cells 500 g for 10 minutes at 4°C in a swing rotor centrifuge.

Discard ALL the supernatant taking care of not loosing the pellet. * Resuspend the pellet in 20 µl of your favorite Lysis buffer containing protease inhibitors.

• Add immediately 25µl of 2X SDS loading buffer and boil the samples at 95°C for 10 min. To avoid condensation and stickiness of DNA, place the tubes immediately in ice . Do not centrifuge!

For separation of proteins, we use the protocol Single Gel system based in Ahn T et al (2001)

Activate gel for detection of proteins using the stainfree system for quantification of proteins present in the sample. These values allow us to do the normalization of the phosphorylation signals after immunodetection.* We use the Bio-Rad Gel Doc™ XR+ Gel Documentation System for the stainfree detection.

• For transfer of proteins to the PVDF membrane, we use the protocol Trans-Blot® Turbo™ Transfer with Home-made buffers

Place the membranes in contact with methanol to activate them.

Block membrane with TBS-T (TBS + 0,075% Tween 20) with 3%BSA for 1 hour at room temperature.

Add the anti-phosphotyrosine antibody in a dilution 1:10000 dilution (Clone 4G10 antibody from Millipore), for 1 hour in PBS with 3% BSA at room temperature.

Wash 4 times, 15 minutes each, with 5 ml TBS-Tween (TBS + 0,075% Tween 20) at room temperature.

Add 1:10000 anti-mouse peroxidase conjugated antibody (Sigma) for 45 minutes in TBS 0,075% Tween 20 .

Wash 4 times 15 minutes with 5 ml TBS 0,075% Tween 20 at room temperature.

Remove ALL WASH BUFFER . Take care to not to let the membrane dry in this step!!!* Add Millipore Immobilon Western (Cat Nr. WBKLS0500) developing solution (1 ml solution A, 1 ml solution B).

• Let the membrane roll with the developing solution for about 1 to 2 min
• Place membrane between to two transparencies.
• Place membrane in Gel documentation machine (Gel Doc™ XR+ Gel Documentation System) and detect the chemoluminiscence

Using the software ImageLab from Bio-Rad, set lanes and bands. Quantify signals using relative values to the control infection.

Using the same image and the image of the Stainfree gel, use the normalization option from the ImageLab software. Multiply the values obtained for the phosphorylation signal with the values obtained for the normalization.