1) Determine the number of cells for injection (ie 5´106 ) to determine the number of plates that
will require trypsinizing (usually a 100% confluent plate of 100mm2 will yield at least 2
injections at 5´106 cells/injection)
2) Trypsinize the number of plates to be counted all at once
3) Collect detached cells in 50 ml conical and spin for 4min at 800 rpm
4) Remove sup and resuspend in 25 ml of SFM for counting
5) Remove three 100 ul aliquots into 3 separate eppendorfs and dilute each 100 ul 1:5 by adding
400u. of SFM, mix well
6) Remove 50 ul of 1:5 dilutions for counting, count each of three dilutions and average the
three numbers
7) Determine the conc. of cells in cells/ml by using the following formula:
Average counts ´ 10,000 ´ dilution factor (5) = #cells/ml
8) Determine the volume required to add to achieve final concentration of cells for injection per
volume to be injected (ie 5´106 cells/ 100 ul injection) by first determining the total number
of cells in the 25 ml suspension by multiplying the conc. of cells in #cells/ml ´ 25 = total
number of cells. Then use the following formula
Total # cells/x volume = 5´106 / 100ul, solve for x = volume to resuspend pellet of cells to
achieve desired final concentration (ie 5´106 cells/ 100 ul)
9) Spin down 50 ml conical for 4 min at 800 rpm
10) Discard sup and resuspend the pellet in the previously determined volume from step #8.
11) Draw up each injection/ mouse in 1 ml syringes in the tissue culture hood prior to going to
the animal facility. Place the separate syringes each containing 100 ul on ice (this step
minimized the possibility of the cells settling after being resuspended thus altering the
concentration of cells.
12) Anesthetize each mouse with isoflorane inhalent just prior to injection. Be careful not to
over anesthetize as the mice will succumb to respiratory depression. Just the right amount is
when they just begin to stop moving, remove them from the source of anesthetic, let them
breath pure air for a few seconds then place their noses just adjacent to the opening of the 50
ml conical duing the injection
13) Inject
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