Desarrollo de la metodologia
PROCESOS MUSICALES ENSAMBLE
Adherent cells were detached from wells using the PBS/ EDTA method and collected by centrifugation. For non-adherent cells, they were collected and centrifuged at maximum 300g
for 5 minutes and the media removed. After collecting cells, pellets were resuspended gently using PBS (-Ca2+,-Mg2+) and washed two times to remove any traces from EDTA in the
solution. After counting and resuspending cells to a concentration 1x106 cells/ml, the detection followed. In the case of detection using fluorescent-labeled primary antibodies, the manufacturer’s recommended concentrations were used and incubation followed for one hour at 4°C. If the use of a non-labeled primary antibody was necessary, it was diluted 1:1000 and incubated one hour at 4°C or 20°C. After washing three times, a secondary fluorescent- labeled antibody was added using a 1:1000 dilution followed by one-hour incubation at 4°C. Two wash steps with PBS removed the unbound antibody. After the cells were fluorescent labeled, they were resuspended in 1 ml of PBS or FACS buffer and stored in ice and darkness until measurement.
2.2.3.6
FACS quantification of integrin β1 levels on the
membrane
AGS cells were splitted in 60 mm plates using the PBS/EDTA method in a dilution 1:4 calculating that 48 hours later they will be 100% confluent. After approx. 30 hours, cells were synchronized overnight. One well was fixed using the “in flagrante” method (see 2.2.3.4). This well represents the initial amount of integrin β1 on the surface after the synchronization. Media containing serum was added, and at the following time points, the same fixation
fixation was for one hour at 4°C store before the addition of antibody. Cells were blocked with an IP blocking buffer overnight at 4°C. Next day cells were washed two times with PBS (+Ca2+, +Mg2+) and supernatant containing primary antibody AIIB2 was added in a concentration 1:100 in blocking buffer for minimum 6 hours at 4°C. Antibody was removed and washed twice with PBS, leaving the reaction in PBS overnight at 4°C for increased specificity. Next day, the washing step was repeated four times, and Alexa488 conjugated anti-
Rat antibody was added in a concentration of 1:2500 for one hour at 4°C. Excess antibody was removed with four washing steps and cells were scrapped from the plate’s surface in 1 ml PBS. The cells were collected and measure their fluorescence intensity in an EPICS® XL- MCL cell sorter (Coulter). Data obtained was analyzed using WinMDI software. By using the mean fluorescence from the population, curves were graphed that describe the amount of integrin β1 present on exposed side of the membrane at the time of fixation in a time dependent way.
Blocking buffer 10% FCS (v/v) in PBS (+Ca2+, +Mg2+).
2.2.3.7
General protocol for Immunostaining
After the fixation of the samples by the “in flagrante” method (see 2.2.3.4), samples on glass coverslides were blocked overnight using 500 µl sterile blocking solution at 4°C. To remove the blocking solution, coverslides were washed three times with 1 ml PBS, each time for 10 minutes. 250 µl of the blocking buffer with primary antibody (1:1000) were added for one hour at 37°C. To remove unbound antibody, five washing steps were repeated. After the primary antibody was removed, 250 µl of blocking solution containing the secondary antibody (1:1000) were added for one hour at 37°C. After one wash step, samples were stored overnight in 500 µl PBS at 4°C. Next morning two washing steps were repeated, and the coverslides were mounted on glass slides using ProLong ® Gold mounting media (Invitrogen). After 24 hours drying at room temperature in darkness, imaging started and/or slides were stored at 4°C.
Blocking solution PBS with 10% FCS
2.2.3.8
HL-60 differentiation
HL-60 cells were counted, and for differentiation 1x106 cells were added to 15 ml RPMI media (Biochrom AG) containing 10% FCS superior (PAA), and 190 µl DMSO. After 6 days
of incubation at 37°C,5% CO2, cells were collected by centrifuging at 300g for five minutes,
then resuspended in media and counted for further work.
2.2.3.9
General Phosphotyrosine assay
Cells were grown to a density of 70-90% depending of the cell type. Bacteria were grown 20- 24 hours on agar plates, resuspended and their OD550 measured. Knowing that a suspension of
H. pylori bacteria OD550 0,1 represents approx. 3 x107 cfu/ml, cells were infected using a
Multiplicity Of Infection (MOI) of 60 ( approx. 60 bacteria per cell). In case of pre-treatment of cells with inhibitors, media was changed and inhibitors added 30 minutes before infection started. Infection was done for one, two or four hours at 37°C and 5% CO2, and stopped by
cold temperature. Supernatants were collected and stored at -20°C for later ELISA detection. Cells were collected in 1 ml ice cold fresh prepared PBS* using a cell scrapper and centrifuged at 500g and 4°C for 10 minutes. Pellets were resuspended in ice-cold RIPA buffer with freshly added protease inhibitors and DNAse I. To prepare the sample for detection of proteins, 2X SDS loading buffer was added and samples boiled in a water bath at 95°C for 10 minutes. Proteins in the samples were separated in a 6% or 8% gel and immunoblotting was done using a mouse polyclonal antibody against phosphorylated proteins PY99 (1:1000) or a mouse monoclonal anti-Phosphotyrosine 4G10 (1:10000). Detection followed using peroxidase conjugated anti-mouse antibodies in a dilution 1:10000.
PBS* PBS (+Ca2+, +Mg2+), 1 µM Sodium- Orthovanadate, 1 µM PMSF, 1 µM Leupeptin, 1 µM Pepstatin, 10µg/ml DNAse I RIPA buffer 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0,25% (w/v)
Sodium deoxycholate, 50 mM Tris HCl pH 7,4, 1 µM Sodium- Orthovanadate, 1 µM PMSF, 1 µM Leupeptin, 1 µM Pepstatin, 10 µg/ml DNAse I.