BMDM Salmonella Replication
The day before .
Inoculate 5 ml of TSB with 1 colony of Salmonella Typhimurium .
Incubate on the wheel at 37C overnight .
Collect BMDM using a cell scraper .
Transfer the cells from one Petri dish to a 50 - ml tube .
Centrifuge 10 min at 1200 RPM .
Remove and discard the supernatant .
Re - suspend the cells using 3 - ml syringe and needle ( start with a 21 or 20 gauge needle and finish with 25 gauge in 2 ml RPMI + 10 % FCS .
Count BMDM using the Coulter Counter or a 1 / 5 dilution with Turks .
( 1.5ml Acetic acid , 48.5 ddH2O and Gentacin Violet – trace ) .
Adjust with fresh cell culture medium the number of cells to a final concentration of 1x10 ^ 6 cells per ml .
Aliquot 500 μL ( 5x10 ^ 5 cells ) per well for a 24 - well plate .
Aliquot 3 mL ( 3x10 ^ 6 cells ) per well for a 6 - well plate .
And 100ul for 96 well plate .
Incubate o / n at 37C , 5 % CO2 .
For LDH , all the samples are in triplicate .
The day of the experiment : Inoculate 50 ml of TSB with 2 ml of the overnight culture of Salmonella .
Grow at 37C with agitation until the OD at 600nm reaches 0.9 ( it takes between 90 - 120 min ) .
Place the bacterial culture on ice and adjust the OD to 0.9 if needed .
In the meantime , prepare all the media which need to be kept at 37C Medium in which to add bacteria PBS for the washes Gentamicin + RPMI + 10 % FCSTitron and PBS for lysis Centrifuge two tubes of 1 ml ( 5 x 10 ^ 8 bact ) of the bacterial culture and resuspend in 1 ml of saline or PBS or RPMI without FCS .
Use Eppendorf tube and centrifuge in the cold room 3 - 4 min at 13000 RPM .
Verify that the BMDM are healthy under the microscope .
Remove the cell culture medium and replace it with RPMI and 10 % FCS Per well : 6 - well plate : 3 ml RPMI + 10 % FCS and 60ul bacteria 24 - well plate : 500 μL RPMI + FCS and 10ul bacteria 96 well plate : 100ul RPMI + 10 % FCS and 2ul bacteria ( 10 μL corresponds approximately to 5x10 ^ 6 bacteria ; MOI of 10 bacteria / cell ) .
For a 6 - well plate , prepare 20 ml of culture medium + 400 μl of washed bacteria .
For a 24 - well plate , prepare 15 ml of culture medium + 300 μl of washed bacteria .
For a 96 well place , prepare 10ml of culture medium and 200ul of washed bacteria .
But for LDH , you only need to prepare enough for the Salmonella wells , not for all 96 wells .
Centrifuge at 500g for 5 minutes at 25C Incubate 45 min at 37C , 5 % CO2 .
During the incubation time , plate the infectious dose 10 ^ - 3 ( 100 μl ) , 10 ^ - 4 ( 100 μl ) and 10 ^ - 5 ( 100 μl ) .
After the incubation period , remove and discard the supernatant .
Wash the cells twice with PBS ( preheated at 37C ) ( prepared before hand ) using : 500 μl for 24 - well plate and 10 ml for 6 - well plate . 100ul for 96 well plate .
Add 500 μL ( 24 - well plate ) or 2 ml ( 6 - well plate ) RPMI 10 % FCS without phenol for LDH ( preheated at 37C ) containing 100 μg / ml gentamicin .
Add an extra triplicate for medium .
For a 24 - well plate , prepare 15 ml of culture medium + 150 μl of gentamicin 10 mg / ml .
For a 6 - well plate , prepare 20 ml of culture medium + 200 μl of of gentamicin 10 mg / ml .
For a 96 well plate , prepare 15ml of culture medium + 150ul of gentamicin at 10mg / ml .
This corresponds to time 0 for LDH .
Start timer for time points and 45 min prior to last time point for max rel .
For all time points : spin plate at 250g for 4min * keep on ice * .
Take 50ul of supernatant for LDH into special LDH plates ( can use multichanel ) and keep on ice .
Take 50ul for ELISA cytokine expression .
Lyse cells with 100ul of PBS - 1 % Titron X - 100 ( prepared in advance ) .
Tranfer 100ul to 2ml push cap tubes for plating .
Dilute 50ul in 450ul for 10 ^ - 1 and 10 ^ - 2 # CFUs / 50μl x 100 for LDH x dilution = CFUs per well .
Continue on for Non LDH experiment : Incubate 1 hour at 37C , 5 % CO2 .
After the incubation period , remove and discard the supernatant .
Wash the cells twice with PBS ( preheated at 37C ) using 500 μl for 24 - well plate ml for 6 - well plate .
Add 500 μL ( 24 - well plate ) or 2 ml ( 6 - well plate ) RPMI 10 % FCS ( preheated at 37C ) containing 10 μg / ml gentamicin .
For a 24 - well plate , prepare 15 ml of culture medium + 15 μl of gentamicin 10 mg / ml .
For a 6 - well plate , prepare 20 ml of culture medium + 20 μl of of gentamicin 10 mg / ml .
Pursue the incubation ( 1 , 2 , and 4 h ) .
Time 0 corresponds to the time just after adding the RPMI containing 10μg / ml gentamicin .
At each time point , collect the supernatants for future cytokine ELISA assays and freeze them at - 80C .
Lyse the cells with 500 μL PBS - 1 % Triton X - 100 and transfer the lysate to 4 - ml push cap tube .
Dilute the lysate ( 10 - 1 to 10 - 3 ) .
Spread 100 μL of 10 - 2 and 10 - 3 dilutions on TSB Petri dishes and incubate overnight .
The day after : Count the CFUs taking into account the volume plated and the dilution .
# CFUs / 100μl x 500 x dilution = CFUs per well
