Jun 07, 2020
Version 2
UT Southwestern - Human Melanoma Metastatic Potential in Mice V.2
This protocol is a draft, published without a DOI.
- Arin Aurora1,
- Sean Morrison1
- 1UT Southwestern, Morrison Lab
- NCI PDMC consortium
Protocol Citation: Arin Aurora, Sean Morrison 2020. UT Southwestern - Human Melanoma Metastatic Potential in Mice. protocols.io https://protocols.io/view/ut-southwestern-human-melanoma-metastatic-potentia-bg9hjz36Version created by Shree Bose
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2020
Last Modified: June 07, 2020
Protocol Integer ID: 37897
Guidelines
This assay is divided in 5 sections:
A.Dissociation of melanomatumors
B.Preparation of cells forinjections
C.Subcutaneous Injection of melanoma cells in NSG mice
D.Mice monitorization
E.Analysis of metastasis atendpoint.
NOTE: FACS protocol for sorting melanoma is separate
Materials
MATERIALS
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
PBS
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
HBSSGibco - Thermo FischerCatalog #14025-092
Leibovitz's L-15 MediumThermo FisherCatalog #11415049
Trypsin (2.5%), no phenol redThermo FisherCatalog #15090046
40um Cell Strainer Fisher ScientificCatalog #22363547
Deoxyribonuclease I from bovine pancreasSigma AldrichCatalog #D4527
EGTA (Ethylene glycol-bis(2-aminoethylether)-NNN′N′-tetraacetic acid)Sigma AldrichCatalog #E4378
PVA (Polyvinyl Alcohol)Millipore SigmaCatalog #P8136
Bovine Serum AlbuminSigma AldrichCatalog #A3912
Lonza™ BioWhittaker™ Water for Cell CultureFischer ScientificCatalog #17724Q
Lonza™ BioWhittaker™ HEPES BufferFischer ScientificCatalog #BW17-737E
STEP MATERIALS
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Deoxyribonuclease I from bovine pancreasSigma AldrichCatalog #D4527
MatrigelCorningCatalog #356231
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJJackson LaboratoryCatalog #005557
Protocol materials
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJThe Jackson LaboratoryCatalog #005557
HBSSGibco - Thermo Fisher ScientificCatalog #14025-092
40um Cell Strainer Fisher ScientificCatalog #22363547
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
PVA (Polyvinyl Alcohol)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8136
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Lonza™ BioWhittaker™ HEPES BufferFischer ScientificCatalog #BW17-737E
Leibovitz's L-15 MediumThermo FisherCatalog #11415049
EGTA (Ethylene glycol-bis(2-aminoethylether)-NNN′N′-tetraacetic acid)Merck MilliporeSigma (Sigma-Aldrich)Catalog #E4378
PBS
Lonza™ BioWhittaker™ Water for Cell CultureFischer ScientificCatalog #17724Q
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3912
MatrigelCorningCatalog #356231
Trypsin (2.5%), no phenol redThermo FisherCatalog #15090046
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
MatrigelCorningCatalog #356231
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJThe Jackson LaboratoryCatalog #005557
DISSOCIATION PREPARATION
DISSOCIATION PREPARATION
Materials:
Surgical dissection tools
Ice
1x HBSS (w/o Ca++ or Mg++) [Gibco #14175]
50 mL Falcon tubes
Disposable pipettes
40 μm cell strainers for Falcon tubes [Fisher #22363547] Cell counter
Make Stock Solutions
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Collagenase Type IV: 2,000 u/mL
make up in HBSS,
Collagenase IV powder stock (Worthington #4189, u/mg varies);
freeze 1 mL aliquots at -20 °C
Deoxyribonuclease I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #D4527
DNase I, type II from bovine Pancreas (10,000 u/mL)
make up in HBSS from powder stock (Sigma#D4527);
freeze 0.5 mL aliquots at -20 °C
CaCl2 (1 Molarity (M) )
Make Digest Media (10 mL volume; make fresh every time):
9 mL HBSS
1 mL 2000 units/mL Collagenase IV Stock Solution (for final 200 units/mL )
50 µL 1 Molarity (M) CaCl2 (for final 5 millimolar (mM) )
50 µL 10000 units/mL DNase (for final 50 units/mL )
Trypsin-EGTA (TEG; 10x stock):
Warm 90 mL PBS
Dissolve 40 mg EGTA and 10 mg PVA into warmed PBS
Add 10 mL 2.5% trypsin (10 mL aliquots stored at –20oC) , stir and cool
pH to 7.4
Filter and store as 10 mL aliquots at –20oC
Make 1x TEG by diluting this stock in HBSS (again, freeze aliquots for later use)
Staining media (for 500ml):
500 mg BSA [Sigma #A3912]
40 mL H2O [Biowhitaker #17724Q]
5 mL 1 Molarity (M) Hepes 7.4 [Biowhittaker #17738E]
5 mL 100x PenStrep [Gibco #15140]
make up to 500 mL with Leibovitz's L15 media
Filter and store at 4 °C
DISSOCIATION OF MELANOMA TUMORS
DISSOCIATION OF MELANOMA TUMORS
Place tumors On ice in HBSS
Note
If tumors are from mice, euthanize by anesthetizing in isoflurane followed by cervical dislocation; take care at dissection to cut off normal tissue
Dissect in petri dish and remove necrotic tissue (pale/whitish tissue located centrally within tumor)
Homogenize in homogenizing tube or chop into small pieces with disposable scalpel
Resuspend in 10 mL of warmed digestion media per 1g of tumor tissue (approximate)
Shake in 37°C water bath for 20min. Shake every 00:05:00 - 00:10:00 by hand.
Wash with HBSS up to 50 mL, centrifuge at 220 x g, 4°C, 00:04:00
Resuspend in 5 mL staining media and filter through 40 μm strainer into new 50 cc tube
Note
From this point on keep the cells in staining medium on ice AT ALL TIMES
For bulk injection, proceed with next section. If sorting first, then proceed with FACS protocol located as a separate file HERE:
Protocol
NAME
UT Southwestern - Staining Melanoma Cells for Flow Cytometry
CREATED BY
Shree Bose
Adjust staining volume and antibody concentration to the number of cells. Concentrations listed are for 5x106 cells in a 50ml reaction. Do not exceed 106 cells/10ml.
- mLin APC: Ter119 (1/100), CD31(1/100), CD45(1/200)
- HLA FITC: HLA-ABC (1/5)
Add antibodies to tube and incubate covered on ice for 00:20:00
Wash 1x with 1 mL staining medium and spin down 220 rpm, 4°C, 00:04:00 . Aspirate supernatant
DAPI staining: resuspend samples in DAPI 1:2000 to gate out dead cells. Stock in made in water (1mg/ml)
Adjust final sample volume to run at 6000 events/sec at maximum and then run. If clogging becomes an issue, then filter through cut pieces of filter
| Xenograft stain (50ml volume) | 1x | 5x | |
| HLA- PE or FITC (1/5) | 10ml | 50ml | |
| mCD45 APC (1/200) | 0.3ml | 1.5ml | |
| mCD31 APC (1/100) | 0.5ml | 2.5ml | |
| mTer119 APC (1/100) | 0.5ml | 2.5ml |
| Human stain (50ml volume) | 1x | 5x | |
| HLA- FITC (1/5) | 10ml | 50ml | |
| ISO PE or P75 PE (1/5) | 10ml | 50ml | |
| Glyc-A APC (1/2000) | 0.025ml | 0.125ml | |
| hu-CD31 APC (1/800) | 0.063ml | 0.315ml | |
| hu-CD45 APC (1/5) | 10ml | 50ml |
PREPARATION OF CELLS FOR INJECTION PREPARATION
PREPARATION OF CELLS FOR INJECTION PREPARATION
Materials
Ice
Eppendorfs
500cc insulin syringes BD #329461 Hemocytometer
Trypan Blue
Stock solutions:
MatrigelCorningCatalog #356231
Thaw overnight in ice, keep On ice at all times
Add 10 mL of cold staining media to bottle
Mix by gently inverting for 00:05:00 aliquot and store at -20 °C
PREPARATION OF CELLS FOR INJECTION
PREPARATION OF CELLS FOR INJECTION
Prepare 10 empty sterile syringes on a Styrofoam box filled up with ice.
Note
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.
Centrifuge sorting tube containing cells at 220 x g, 4°C, 00:04:00
Resuspend pelleted cells in a small volume that will allow accurate counting.
Count cells in hemocytometer with Trypan blue to determine concentration of cells cells.
Note
If sorted, may lose roughly 20-30%
For 10 injections, prepare 700 µL of cells at 100 cells/50 uL in 25% matrigel.
Prepare a cell suspension of 1400 sorted cells into 350 µL of cold SM.
Add to above 350 µL of 50% matrigel solution and resuspend with pipet up and down 5 times to get to an even solution of cells.
Load 10 syringes with 50 µL of the cell suspension each and maintain loaded syringes on ice at all times.
Note
Take leftover cells on ice in case you need extra in mouse room
INJECTION PREPARATAION
INJECTION PREPARATAION
Materials:
Syringes loAded with 50 l of sorted melanoma cells in matrigel
50 ml conical tube with holes in tip to allow mouse ventilation.
Mice injection sheet with records (see example attached)
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJThe Jackson LaboratoryCatalog #005557
SUBCUTANEOUS INJECTION OF MELANOMA CELLS IN NSG MICE
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)
Write your name, the injected tumor lD, date and “metastasis assay” on the cage card.
Prepare a clean cage where you will transfer mouse by mouse as soon as the injection is done.
Choose one mouse from uninjected cage, immobilize mouse and ear tag (see guide).
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.
Transfer mouse to the clean cage and proceed with the next mouse.
MICE MONITORIZATION
MICE MONITORIZATION
Mice will be monitored every 15 days by palpation at site of injection
Note
Once tumors become measurable, measure and write down the biggest diameter weekly