Xenograft Tumor Assay Protocol

1) Determine the number of cells for injection (ie 5´106 ) to determine the number of plates thatwill require trypsinizing (usually a 100% confluent plate of 100mm2 will yield at least 2injections at 5´106 cells/injection)2) Trypsinize the number of plates to be counted all at once3) Collect detached cells in 50 ml conical and spin for 4min at 800 rpm4) Remove sup and resuspend in 25 ml of SFM for counting5) Remove three 100 ul aliquots into 3 separate eppendorfs and dilute each 100 ul 1:5 by adding400u. of SFM, mix well6) Remove 50 ul of 1:5 dilutions for counting, count each of three dilutions and average thethree numbers7) Determine the conc. of cells in cells/ml by using the following formula:Average counts ´ 10,000 ´ dilution factor (5) = #cells/ml8) Determine the volume required to add to achieve final concentration of cells for injection pervolume to be injected (ie 5´106 cells/ 100 ul injection) by first determining the total numberof cells in the 25 ml suspension by multiplying the conc. of cells in #cells/ml ´ 25 = totalnumber of cells. Then use the following formulaTotal # cells/x volume = 5´106 / 100ul, solve for x = volume to resuspend pellet of cells toachieve desired final concentration (ie 5´106 cells/ 100 ul)9) Spin down 50 ml conical for 4 min at 800 rpm10) 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 tothe animal facility. Place the separate syringes each containing 100 ul on ice (this stepminimized the possibility of the cells settling after being resuspended thus altering theconcentration of cells.12) Anesthetize each mouse with isoflorane inhalent just prior to injection. Be careful not toover anesthetize as the mice will succumb to respiratory depression. Just the right amount iswhen they just begin to stop moving, remove them from the source of anesthetic, let thembreath pure air for a few seconds then place their noses just adjacent to the opening of the 50ml conical duing the injection13) Inject