protocol Elisa macrophages
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