Procedure
A. PD study set-up
1. Determine the logistics of your PD study including the number of mice required, treatment (use of single agent/combination), treatment dose, schedule (one day/round/week), and treatment time before harvest before you commence your experiment
2. Inoculate mice (SWP 0090) or source vehicle-treated mice from a prior efficacy study and allow the mice to reach ~60-80% engraftment in the peripheral blood. Mouse spleens can be palpated to confirm that they are highly engrafted
3. Separate the mice into vehicle and drug treatment groups (you may need to rearrage cages and re tag mice (SWP0081)). Each group should consist of 3-4 mice per drug or time-point.
4. Weigh all mice and record their initial weights (SWP 0089 …show more content…
Bleed all mice (SWP 0082) to obtain day 0 peripheral blood engraftment values by flow cytometry (SWP 0093)
6. Treat mice according to your chosen schedule. For detailed drug administration techniques, see appropriate SWP (SWP 0083/SWP 0084/SWP0087/SWP 0088).
NOTE: The mice will need to be closely monitored following treatment as they will be more sensitive to drug treatment as they are highly engrafted. If the mouse becomes moribund it will need to be euthanised (SWP 0020).
B. Collection of samples and tissues
1. Book one hood in the Animal Facility to harvest mouse tissues and one in the Tissue Culture Suite for processing of tissues. If your cells do not need to remain sterile, the latter can be done on your bench
2. Pre-warm media in a 37°C waterbath
3. If you require viable cells, ensure that there are enough cool boys at room temperature for the harvest
4. Weigh and bleed mice to obtain final values
5. Humanely euthanise mice (SWP …show more content…
Remove the required organs (spleen, bone) to be harvested; organs should be removed with sterile surgical forceps and scissors, then placed in a sterile specimen jar containing media. Collect a small portion of each of these organs for flow analysis to determine leukemic infiltration
8. To harvest viable cells, follow the protocol in SWP 1030. For cells that do not need to remain viable (e.g. DNA/RNA/protein analysis), follow the same protocol, however, instead of freezing the cells in FCS+DMSO, aliquot the cell suspension into ependorph tubes at ~20M cells/tubes. Centrifuge at 1500 rpm for 5 min at RT and remove supernatant. Snap freeze in liquid nitrogen and store at -80°C until required for analysis.
F. Shutdown/Cleanup
1. Throw all solid waste into the appropriate bin (PC2 or cytotoxic).
2. Wipe down the surface of the hood with F10SC and then 80% ethanol
3. Shut the hood down and turn on the UV
A. PD study set-up
1. Determine the logistics of your PD study including the number of mice required, treatment (use of single agent/combination), treatment dose, schedule (one day/round/week), and treatment time before harvest before you commence your experiment
2. Inoculate mice (SWP 0090) or source vehicle-treated mice from a prior efficacy study and allow the mice to reach ~60-80% engraftment in the peripheral blood. Mouse spleens can be palpated to confirm that they are highly engrafted
3. Separate the mice into vehicle and drug treatment groups (you may need to rearrage cages and re tag mice (SWP0081)). Each group should consist of 3-4 mice per drug or time-point.
4. Weigh all mice and record their initial weights (SWP 0089 …show more content…
Bleed all mice (SWP 0082) to obtain day 0 peripheral blood engraftment values by flow cytometry (SWP 0093)
6. Treat mice according to your chosen schedule. For detailed drug administration techniques, see appropriate SWP (SWP 0083/SWP 0084/SWP0087/SWP 0088).
NOTE: The mice will need to be closely monitored following treatment as they will be more sensitive to drug treatment as they are highly engrafted. If the mouse becomes moribund it will need to be euthanised (SWP 0020).
B. Collection of samples and tissues
1. Book one hood in the Animal Facility to harvest mouse tissues and one in the Tissue Culture Suite for processing of tissues. If your cells do not need to remain sterile, the latter can be done on your bench
2. Pre-warm media in a 37°C waterbath
3. If you require viable cells, ensure that there are enough cool boys at room temperature for the harvest
4. Weigh and bleed mice to obtain final values
5. Humanely euthanise mice (SWP …show more content…
Remove the required organs (spleen, bone) to be harvested; organs should be removed with sterile surgical forceps and scissors, then placed in a sterile specimen jar containing media. Collect a small portion of each of these organs for flow analysis to determine leukemic infiltration
8. To harvest viable cells, follow the protocol in SWP 1030. For cells that do not need to remain viable (e.g. DNA/RNA/protein analysis), follow the same protocol, however, instead of freezing the cells in FCS+DMSO, aliquot the cell suspension into ependorph tubes at ~20M cells/tubes. Centrifuge at 1500 rpm for 5 min at RT and remove supernatant. Snap freeze in liquid nitrogen and store at -80°C until required for analysis.
F. Shutdown/Cleanup
1. Throw all solid waste into the appropriate bin (PC2 or cytotoxic).
2. Wipe down the surface of the hood with F10SC and then 80% ethanol
3. Shut the hood down and turn on the UV