TITLE: UT Southwestern - Human Melanoma Metastatic Potential in Mice
AUTHORS: Arin Aurora, Sean Morrison

[STEPS]
?. [DISSOCIATION PREPARATION]
Materials: Surgical dissection toolsIce1x HBSS (w/o Ca++ or Mg++) [Gibco #14175]50 mL Falcon tubesDisposable pipettes40 μm cell strainers for Falcon tubes [Fisher #22363547] Cell counter Make Stock SolutionsCollagenase Type IV: 2,000 u/mL make up in HBSS, Collagenase IV powder stock (Worthington #4189, u/mg varies); freeze 1 mL aliquots at DNase I, type II from bovine Pancreas (10,000 u/mL)make up in HBSS from powder stock (Sigma#D4527); freeze aliquots at CaCl2 ()
-20 °C
0.5 mL
-20 °C
?. [DISSOCIATION PREPARATION]
Make Digest Media (10 mL volume; make fresh every time):   HBSS Collagenase IV Stock Solution (for final  ) CaCl2  (for final  ) DNase (for final )
9 mL
1 mL
50 µl
50 µl
?. [DISSOCIATION PREPARATION]
Trypsin-EGTA (TEG; 10x stock):Warm  PBSDissolve  EGTA and PVA into warmed PBSAdd  2.5% trypsin (10 mL aliquots stored at –20oC) , stir and cool pH to Filter and store as 10 mL aliquots at –20oC Make 1x TEG by diluting this stock in HBSS (again, freeze aliquots for later use)
90 mL
40 mg
10 mg
10 mL
?. [DISSOCIATION PREPARATION]
Staining media (for 500ml): BSA [Sigma #A3912] H2O [Biowhitaker #17724Q] Hepes [Biowhittaker #17738E]  100x PenStrep [Gibco #15140]make up to  with Leibovitz's L15 media Filter and store at
500 mg
40 mL
5 mL
5 mL
500 mL
4 °C
?. [DISSOCIATION OF MELANOMA TUMORS]
Place tumors  in HBSS
on ice
If tumors are from mice, euthanize by anesthetizing in isoflurane followed by cervical dislocation; take care at dissection to cut off normal tissue
?. [DISSOCIATION OF MELANOMA TUMORS]
Dissect in petri dish and remove necrotic tissue (pale/whitish tissue located centrally within tumor)
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Homogenize in homogenizing tube or chop into small pieces with disposable scalpel
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Resuspend in 10 mL of warmed digestion media per 1g of tumor tissue (approximate)
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Shake in 37°C water bath for 20min. Shake every  -  by hand.
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Wash with HBSS up to 50 mL, centrifuge at
Centrifuge: 220 34, 4 min, 4 10
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Resuspend in 5 mL staining media and filter through 40 μm strainer into new 50 cc tube
From this point on keep the cells in staining medium on ice AT ALL TIMES
?. [DISSOCIATION OF MELANOMA TUMORS]
For bulk injection, proceed with next section. If sorting first, then proceed with FACS protocol located as a separate file HERE:
?. [PREPARATION OF CELLS FOR INJECTION PREPARATION]
MaterialsIceEppendorfs500cc insulin syringes BD #329461 HemocytometerTrypan Blue Stock solutions:Thaw overnight in ice, keep  at all timesAdd  of cold staining media to bottleMix by gently inverting for  aliquot and store at
on ice
10 mL
-20 °C
?. [PREPARATION OF CELLS FOR INJECTION]
Prepare 10 empty sterile syringes on a Styrofoam box filled up with ice.
You will need to thaw the right amount of 50% matrigel 2h before preparing cells. Matrigel should be kept on ice at ALL TIMES to be thawed.
?. [PREPARATION OF CELLS FOR INJECTION]
Centrifuge sorting tube containing cells at
Centrifuge: 220 34, 4 min, 4 10
?. [PREPARATION OF CELLS FOR INJECTION]
Resuspend pelleted cells in a small volume that will allow accurate counting.
?. [PREPARATION OF CELLS FOR INJECTION]
Count cells in hemocytometer with Trypan blue to determine concentration of cells cells.
If sorted, may lose roughly 20-30%
?. [PREPARATION OF CELLS FOR INJECTION]
For 10 injections, prepare  of cells at 100 cells/50 uL in 25% matrigel.
700 µl
?. [PREPARATION OF CELLS FOR INJECTION]
Prepare a cell suspension of 1400 sorted cells into   of cold SM.
350 µl
?. [PREPARATION OF CELLS FOR INJECTION]
Add to above   of 50% matrigel solution and resuspend with pipet up and down 5 times to get to an even solution of cells.
350 µl
?. [PREPARATION OF CELLS FOR INJECTION]
Load 10 syringes with  of the cell suspension each and maintain loaded syringes on ice at all times.
50 µl
Take leftover cells on ice in case you need extra in mouse room
?. [INJECTION PREPARATAION]
Materials: Syringes loAded with 50 l of sorted melanoma cells in matrigel50 ml conical tube with holes in tip to allow mouse ventilation.Mice injection sheet with records (see example attached)
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Choose a cage with 5 mice (at least 4 weeks old) and write down information on mice injection sheet (cage ID, gender and date of birth)
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Write your name, the injected tumor lD, date and “metastasis assay” on the cage card.
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Prepare a clean cage where you will transfer mouse by mouse as soon as the injection is done.
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Choose one mouse from uninjected cage, immobilize mouse and ear tag (see guide).
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Restrain mouse in the conical 50ml tube and inject slowly subcutaneously in right flank. After injecting press injected site with your fingers for a few seconds.
?. [SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE]
Transfer mouse to the clean cage and proceed with the next mouse.
?. [MICE MONITORIZATION]
Mice will be monitored every 15 days by palpation at site of injection
Once tumors become measurable, measure and write down the biggest diameter weekly