Part 1: Retronectin Coating
Step 1: Take non-TC treated 24-well polystyrene plate and add 300 microliters per well of Retronectin diluted to 100 microgram/mL in PBS. Note # of wells = # of donor mice x 4.
Step 2: Place in fridge overnight wrapped in saran wrap. Note can keep for several days.
Part 2: LSK Isolation
Step 1: Sacrifice mouse and pin down at the neck.
Step 2: Cut skin down legs fully. Pull leg until you hear a pop. Cut away muscle and pull off tibia/femur and place in RPMI 10% FBS on ice.
Step 3: Remove pelvic bones and place in RPMI 10% FBS on ice.
Step 4: Remove spine and cut into half inch sizes pieces and place in RPMI 10% FBS on ice.
Step 5: Clean excess tissue from the bones using a Kim wipe and scissors. Place clean bones in 50 mL conical with RPMI 10% FBS on ice.
Step 6: Clean mortar and pestle with 70% ethanol and air dry. Note: treat mortar and pestle and all subsequent steps with sterile technique.
Step 7: Dump bones in mortar. Wash 2x with 70% ethanol.
Step 8: Wash 2x with RPMI 10% FBS.
Step 9: Add 10 mL of RPMI 10% FBS to mortar. Grind up bones with pestle until white.
Step 10: Move the supernatant (leaving bones in mortar) to a 50 mL conical with 70 micron filter top.
Step 11: Repeat steps 9 and 10.
Step 12: Bring up volume to 50 mL with RPMI 10% FBS. Spin conical at 400g for 8 minutes at 4°C.
Step 13: Remove supernatant and resuspend the pellet in 1 mL of ACK lysis buffer per mouse (4 donors = 4 mL). Incubate for 2 minutes at room temperature.
Step 14: Neutralize the ACK lysis buffer with 50 mL RPMI 10% FBS. Pipette through a 70 micron filter into a new 50 mL conical.
Step 15: Spin conical at 400g for 8 minutes at 4°C.
Step 16: Remove the supernatant. Resuspend the pellet in 2 mL of MACS buffer (500 mL PBS + 5 mL FBS + 2 mM EDTA).
Step 17: Add 120 microliters of CD117 microbes per mouse (4 mice = 480 microliter) and mix. Incubate on ice for 20 minutes.
Step 18: Bring up volume to 50 mL with MACS buffer. Spin conical at 400g for 8 minutes at 4°C.
Step 19: During the spin setup Miltenyi MACS magnets with 3 mL LS columns (2 columns per mouse). Equilibrate with 3 mL of MACS buffer.
Step 20: Remove supernatant and resuspend cells in 4 mL of MACS buffer and pipette through a 70 micron filter into a new 50 mL conical. Add MACS buffer such that the final volume in mL = 4 x # of mice (16 mL for 4 mice).
Step 21: Pipette 2 mL into each column and allow the unbound cells to drip into a waste reservoir.
Step 22: Add 3 mL of MACS buffer to each column and allow the wash to drip into a waste reservoir. Repeat for a total of 2 washes.
Step 23: Add 5 mL of MACS buffer to each column and remove from the magnet. Place the column over a 50 mL collection tube and using the plunger provided with LS columns forcefully plunge the 5 mL into the collection tube. Note: do 4 columns at a time (maximum) to ensure the 5 mL elution does not drip through into the waste reservoir while plunging other columns.
Step 24: Bring up volume to 50 mL with MACS buffer. Spin conical at 400g for 8 minutes at 4°C.
Step 25: Remove supernatant and resuspend cell pellet in 1 mL of antibody stain per 4 mice. Antibody stain is composed of 1 mL of MACS and 10 microliter each of the antibodies: Ter119-PE, CD3e-PE, CD5-PE, B220-PE, CD11b-PE, Gr1-PE, cKit-APC, and Sca1-BV421. Cover with tinfoil and stain for 30 minutes on ice.
Step 26: Bring up volume to 50 mL with MACS buffer. Spin conical at 400g for 8 minutes at 4°C.
Step 27: Remove supernatant and resuspend pellet in 0.5 mL per mouse. Pipette through a Polystyrene filter top tube slowly. Wash the top of the filter top tube with an additional 0.5 mL per mouse. Remove the filter top with a solid top and place cells on ice.
Step 28: Prepare sort collection tubes. Place 500 microliter of SFEM supplemented with
100 ng/mL of the following cytokines: SCF, TPO, Flt3-Ligand, and IL-7. Keep collection tubes on ice.
Step 29: Ensure sorter is setup properly (Accudrop, test sort, and laser delay). Load sample onto sorter and draw the following sequential gates (plot attached in images): Live cells (based on FSC vs SSC) -> Singlet cells (based on FSA vs FSH) -> Lineage– cKit+ (based on Lineage-PE vs cKit-APC) -> cKit+ Sca1+ (based on cKit-APC+ Sca1+).See figure in Figures section.
Step 30: Sort your Lineage– cKit+ Sca1+ (LSK) cells into your collection tube until you run out of sample.
Step 31: Once sort is complete spin collection tube at 400g for 10 minutes at 4°C.
Step 32: Remove supernatant and resuspend pellet in SFEM supplemented with SCF, TPO, Flt3-Ligand, and IL-7 (as above). Resuspend with 200 microliter per 100K sorter counted cells.
Step 33: Plate the resuspended cells in a 96 well flat bottom plate at 200 microliter per well. Place in a standard 37°C tissue culture incubator overnight.
Day 2: LSK Transduction
Step 1: Fast temp a centrifuge to 37°C.
Step 2: Look at plated LSK and check for contamination (cloudy media, visible bacteria under microscope). If contaminated will need to repeat the experiment.
Step 3: Aspirate liquid from Retronectin coated plate and add 300 microliter PBS to each well pipetting up and down to wash the well. Then aspirate liquid again.
Step 4: Pipette the 200 microliter of plated LSK into the Retronectin coated plate wells (1 96 well goes into 1 24 well).
Step 5: Add titered lentivirus on top of LSK into the 24 well plate. Volume of lentivirus is calculated as: Volume in mL = 100,000 LSK ** MOI of 30/ Concentration of lentivirus in viral particles per mL. For example 100,000 LSK ** MOI of 30/10,000,000 particles per mL = .333 mL.
Step 6: Add SFEM supplemented with cytokines (as above) such that the final volume in the well is 400 microliters. SFEM Volume = 400 microliter - 200 microliter (LSK) - lentivirus volume in microliter. Pipette up and down in the well gently to mix.
Step 7: Prepare a balance plate and spin the plate at 650g for 1.5 hours at 37°C with an acceleration of 2 and a brake of 1.
Step 8: When spin is complete move the plate to a 37°C tissue culture incubator for 1 hour.
Step 9: Pre-warm SFEM supplemented with cytokines (as above) to 37°C. Prepare 500 microliter * the # of wells in the plate you are using.
Step 10: After the hour is complete add 500 microliters prewarmed stem cell media (with cytokines) on top of the LSK. Place back in the incubator overnight.
Day 3: LSK Transfer to Irradiated Recipients
Step 1: Look at plated LSK and check for contamination (cloudy media, visible bacteria under microscope). If contaminated will need to repeat the experiment.
Step 2: Irradiate recipient mice with 600 rads in a suitable irradiator.
Step 3: Dose the mice with 200 mg sulfamethoxazole and 40 mg trimethoprim per 250 mL of drinking water. Continue this for the next 2 weeks.
Step 4: Three and a half hours after the first irradiation pipette stem cells into a 50 mL conical and bring up the volume to 50 mL with PBS.
Step 5: Spin conical at 400g for 8 minutes at 4°C.
Step 6: Remove supernatant and resuspend in PBS in a volume equal to (200 microliter * the number of wells that had LSK) + 300 microliter. Place this in a suitable tube on ice.
Step 7: Irradiate recipient mice with 600 rads in a suitable irradiator.
Step 8: Inject the LSK intravenously (200 microliter per mouse). For tail vein injections it is recommended to heat the mouse with a heat lamp before injection, place in a restrainer, and wipe the injection site with ethanol. These steps increase your chance of a successful injection.
Step 9: After injecting the mice place back into their cage and allow to reconstitute over the next 8 weeks.
Days 4-17: Antibiotic Treatment
Step 1: Dose the mice with 200 mg sulfamethoxazole and 40 mg trimethoprim per 250 mL of drinking water. Continue this, changing the drinking water as needed, for a total of 2 weeks post LSK injection.