obtain Peritoneal macrophages from BALB/c Mice.

1) inject mice with 3 ml thioglycolate broth (encourages macrophages to come to cavity)

2) wait 4 days

3) inject PBS

 

Split cells into control group and LPS stimulated group

1) obtain 24 well culture plate and add cells as below:

control (Mq only)                                                                  Mq + LPS

2 x 105 cells/ml     2 x 105 cells/ml + LPS (1 ulg/ml)    
2 x 105 cells/ml     2 x 105 cells/ml + LPS (1 ulg/ml)    
1 x 106 cells/ml     1 x 106 cells/ml + LPS (1 ulg/ml)    
1 x 106 cells/ml     1 x 106 cells/ml + LPS (1 ulg/ml)    

 

[cells]  cells in tubes are already at 1 X 106 cells/ml, so take 1 ml and put in appropriate wells to give this concentration. 2 X 105 cells/ml is 5 times less than 1 X 106 cells/ml, since 2X 5 = 10. So take 200 ul of cells and add 800 ul of medium for this concentration.

LPS comes in a stock 1mg/ml. We want final concentration 1 ug/ml. How many ul from stock should we add to wells?

(CI VI = C2 V2)     First, get the same units. ( 1 mg/ml) (1000 ug/mg) = (1000 ug/ml)

(1000 ug/ml) VI =  (1 ug/ml) (1000 ul)

VI = 1 ul 

 

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