Mouse BMDM
Put 2 mL media per well in 6 - wells plate ( 1 well per 2 marrows or per animal ) .
Clean femur as much as possible in a petri with a little bit of phenol red free media or PBS ( the most important part is the middle ) .
Cut off both ends of the femur to expose the marrow .
Fill syringe up with the 2ml of media , insert into marrow and flush into the 6 well plate until the bone is clean ( goes from pink to white ) .
Pass the media through the syringe 1 - 2 times ( Start with 20G , then 26G ) .
Transfer the cells to a 50 ml tube .
Prepare RBC lysis solution ( proportions in cell culture cabinet ) – Or purchase from Sigma
Add 5ml per marrow .
Place on ice for 10min excatly .
Centrifuge for 10min at 4°C 400g ( ~ RCF , or 1200 rpm ) .
Discard supernatant .
Add 5ml CSF and 12ml of RPMI Medium ( total volume is 15 ml ) – Do not let cells dry , add medium immediately . ( Or 9ml RMPI medium and 3ml CSF )
Transfer the 15 mL cell suspension to a 100 mm polystyrene tissue culture petris ( PrimariaTM , Becton Dickinson Labware ) for 24 hrs .
Transfer non - adherent cells after 24 hrs to fresh polystyrene petris or non adherent flasks ( Green - Fisher ) .
Cultures are grown for 6 days with 15 % ( v / v ) L - 929 cell - conditioned medium as a source of M - CSF .
Add fresh CSF every 3 days . ( 15 % of 15ml – ( about 3ml )
The batch made by Kyoko and Rabi ais at 30 % , so use 5ml .
After 6 days , collect the cell with a policeman ( cell scraper 25 cm , Sarstedt # 83,1830 ) .
Centrifuged for 10min 400 x g . Discard supernatant and resuspend pellet in 2ml of RPMI 10 % FBS without Pen / Strep Phenol free for LDH .
Flush media through 25 , 27 , and 30 G needles to separate aggregates .
Count cells to final concentration of 1 x 106 cells / ml .
To count cells , dilute 1 / 5 in Turks .
Aliquot 500 µL ( 5x105 cellules ) per well in 24 - wells plate , 100ul for 96 well plate .
Incubate o / n , 5 % CO2 before infection .
