Xenograft Dissociation and Mouse Cell Depletion Using Miltenyi Equipment

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1 1. Purpose The purpose of SOP 2.7 is to provide details on how to obtain a single cell suspension from xenografts using the Miltenyi gentlemacs Octo dissociator and then mouse cell depletion on suspension using the MultiMACS 24Cell separator. 2. Scope SOP 2.7 is intended to cover all resources, personnel and equipment in the BCR laboratory 3. Materials No. Name Description Storage Location 1.0 Scalpels Size 10 blade; blue handle Necropsy Room Top or 10 cm petri dish Stock room ( S-A) 3.0 PBS Phosphate buffered saline 1X, Gibco# BSA Bovine Serum Albumin, Roche cat# Cold Storage ( C) or Fridge 7 Necropsy room Fridge 7 Necropsy room um cell strainer Purple - Miltenyi cat# Necropsy room 374C bottom um cell strainer Yellow - Miltenyi cat# Necropsy room 374C 3 rd 7.0 RPMI Gibco cat# Cold storage ( C) 8.0 C-tubes Purple blender cap Miltenyi cat# Human tumor digestion kit Miltenyi cat# Enzymes H, R and A Necropsy room 374C 2 nd Fridge 7 or frozen stock Freezer 7 Necropsy Room 10.0 Mouse cell depletion kit Miltenyi cat# Fridge 7 Necropsy room ml conical tubes BD Stock Room ( C-A) 12.0 LS columns Miltenyi cat# Necropsy room 374C 3 rd Drawer L of Hood 4. Reagent Preparation Enzyme H, R, A should be prepared in aliquots stored in labeled boxes in freezer 7. If there are no aliquots available, please see protocol Tumor Dissociation Kit Enzyme Preparation Thaw aliquots on ice; for each C-tube (<1g tumor), thaw one aliquot of H, R, and A Cell Separation Buffer PB Buffer : Prepare with PBS (Phosphate Buffered Saline) and 0.5% BSA (Bovine Serum Albumin) For 100mL of Buffer, dissolve 500mg BSA in 100mL PBS Once dissolved, sterile filter mixture and store in at 4 C Page 1

2 5. XENOGRAFT DISSOCIATION USING MILTENYI TUMOR DISSOCIATOR Store tissue/cells on ice between each step and work quickly to avoid cell degradation Remove and weigh tumor(s) Remove necrotic tissue where possible in center of tumor. Total weight tissue should not exceed 1g for each C-Tube In each C-tube to make enzyme mix add: ml RPMI or DMEM uL reagent H, 100uL reagent R, 25uL reagent In a 6 or 10 cm petri dish; mince tumor into <2mm x 2mm pieces Use scalpel or razor blade to transfer tumor slurry to C tube. Make sure lid is completely closed; you should feel it click into place Turn on GentleMACS Octo-Dissociator Place C-tube upside down on the machine, clicking the tube into place Put on heater jacket (yellow) Select 37C_h_TDK_1, 2 or 3 program (1 for soft, 2 for med & 3 for hard tumors); program runs ~1hr You should have a cloudy suspension with minimal chunks when done If larger chunks still remain: Remove and invert the c-tube and allow it to settle for ~5 min Remove 4ml of supernatant leaving ~1mL media and tumor chunks Put supernatant in fresh tube and place C-tube back on dissociator Run program m_imptumor_ Combine the resulting cell suspension with the previously removed supernatant and re-suspend Apply sample to 70 um MACS SmartStrainer (purple strainer) Rinse filter with 10mL RPMI or DMEM Rinse C-tube AND lid with 10mL media and filter into same 50mL tube Centrifuge at g x 10min at 4 C Remove cloudy supernatant Re-suspend pellet with 10mL media and apply sample to 30um MACS SmartStrainer (yellow filter) Wash original 50mL tube with 10mL media and filter thru 30um filter into same tube Measure volume (vol. should be ~20mL) Count cells on Luna -> 18uL cells + 2uL AO/PI. *save images if needed Tumor Vol (ml) Dilution Boxes Size Viability # Live # Dead Conc/Live (x10 6 /ml) Total (x10 6 ) Page 2

3 SOP BCR MOUSE CELL DEPLETION USING MILTENYI MULTIMACS CELL SEPARATOR Do not exceed 5 x 10 7 total cells per LS column. For samples > 5 x 10 7 split into multiple LS Columns. *Note: If using a different type of column please see user manual for maximum cell capacity If cells appear to be adhering to each other or the sample appears slimy after digestion: Centrifuge sample at 300g x 10 min Aspirate supernatant Resuspend pellet in 5mL PRMI, DMEM or PB Buffer (with no additives) Add 200U/mL DNase and incubate for 5 min at Room Temp Rinse with 20mL RPMI or DMEM (w/ Serum) Centrifuge at 300g x 5 min Split samples into separate 50mL tubes now if the cell count is >50 million, so that all samples have 50 million cells each Centrifuge at g for 10 min at 4 C Asperate supernatant Add Buffer using the following graph for appropriate volumes based on cell numbers (result should be a Add Mouse Cell Depletion Kit beads using the following graph for appropriate volumes based on cell numbers *Example: Resuspend cell pellet in 80uL of Buffer per 1 x 10 7 total cells (including RBCs). Then add 20uL Mouse Cell Depletion Cocktail per 1 x 10 7 total cells (including RBCs) 1 x x x x x 10 7 Buffer (ul) MDCK (ul) Incubate 15 min at 4 C (Refrigerate - do NOT use ice) During Incubation, turn on MulitMACS Cell24 Separator. Select MCDK Program and follow prompts. *Always wait until the column reservoir is empty before proceeding to the next step Insert appropriate number of columns based on # of tubes you have after cell counting Add equilibrium buffer = 3 x 1ml PB buffer to each column (With deep well waste plate in place) Tip touch by pushing back 1 to 2 times on metal base plate under the columns Remove deep well waste plate and discard waste After incubation, use table below to determine volume of PB Buffer to add to Mouse Bead/Cell Mixture. *Example: Final volume for sample with 1x10 7 should be 500uL. 1 x x x x x 10 7 Buffer (ul) Place 5mLTube Rack holder on black base plate with FACS tubes in place under each column (remove caps) Apply cells/beads/buffer sample solution to LS columns Run-through is unlabeled cells representing enriched human tumor cells. Put samples on ice Place new tubes under columns and wash with 2 x 1mL PB Buffer Page 3

4 Combine wash run-through (cells will represent enriched human tumor cells) with unlabeled cells from previous step There should be ~ mL of cells per 5mL FACS tube depending on the number of cells you started with Centrifuge at g for 5 min at 4C Resuspend in appropriate media for downstream analysis Count cells on Luna -> 18uL cells + 2uL AO/PI. *save images if needed Tumor Vol (ml) Dilution Boxes Size Viability # Live # Dead Conc/Live (x10 6 /ml) Total (x10 6 ) Mouse Side (left/right) Size (mm) Weight (mg) # C-tubes Volume post Digestion (ml) Size (um) Conc (x10 6 /ml) Total (x10 6 ) MCDK (ul) PBS/BSA (ml) # LS columns Volume post Depletion (ml) Size (um) Conc (x10 6 /ml) Total (x10 6 ) Cell yield Page 4

5 7. Applicable References SOP 3.3 Cell Freeze andthaw SOP 4.30 Preparation of Miltenyi GentleMACS dissociation enzymes SOP 4.31 Preparation of PB buffer for mouse cell depletion Miltenyi Biotec Tumor Dissociation Kit (human) protocol (order number Miltenyi Biotec Mouse Cell Depletion Kit Protocol 8. Change Description Revision Date Reference Description of Change 8.1 9/21/15 TL 8.2 4/7/2017 KH Added viability column to chart in steps 4.15 and 4.34; added dead cell removal step Reworded all, added descriptions, changed max cell counts, added DNase protocol option, added references, changed tables Page 5