Materials and Reagents

Tumor cells
Note: Tumor sample is harvested from pet dogs during brain surgery.
BD Matrigel^TM (BD Bioscience, catalog number: 354230 )
DMEM/F12 Medium with L-glutamine (Life Technologies, Gibco^®, catalog number: 11320-033 )
50x B-27 supplement without vitamin A (Life Technologies, Gibco^®, catalog number: 12587-010 )
100x N2 supplement (Life Technologies, Gibco^®, catalog number: 17502-048 )
Recombinant human Epidermal Growth Factor (Pepro Tech, catalog number: AF-100-15 )
Recombinant human fibroblast growth factor-basic (Pepro Tech, catalog number: 100-18B )
50x Penicillin-streptomycin solution (Mediatech, Cellgro^®, catalog number: 30-001-CI )
Dispase (BD Bioscience, catalog number: 354235 )
1x Phosphate-Buffered Saline (Mediatech, Cellgro^®, catalog number: 21-040-CV )
MACS BSA stock solution (10% BSA) (Miltenyi Biotec, catalog number: 130-091-376 )
1x TrypLE^TM Express (Life Technologies, Gibco^®, catalog number: 12605-010 )
0.4% Trypan Blue Solution (Life Technologies, Gibco^®, catalog number: 15250-061 )
Goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate (American Qualex, catalog number: F145FN )
Neural Stem Cell (NSC) medium (see Recipes)
Matrigel Coating solution (see Recipes)
rh-EGF stock solution (see Recipes)
rh-FGF basic stock solution (see Recipes)

Equipment

10 mm culture plates (Sigma-Aldrich, Corning^® Costar^®, catalog number: CLS430165 )
Scalpel blade
100 μm filter (Corning Incorporated, catalog number: 352360 )
BD FACS tubes (12 x75 mm) (BD Biosciences, Falcon^®, catalog number: 352235 )
15 ml tubes (BD Biosciences, Falcon^®, catalog number: 352096 )
Culture dish (100 x 20 mm) (Sarstedt AG, catalog number: 83.1802 )
1.5 ml Eppendorf microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661-500EA )
Neubauer hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
Incubator (5% CO₂, 5% O₂, 37 °C) (BioSpherix)
Shaker table (Eppendorf, New Brunswick Excella^® E25 incubator shaker)
Centrifuge (Thermo Fisher Scientific, model: ST40R )
Microcentrifuge (Thermo Fisher Scientific, model: MicroCL 17 )
BD FACSCanto three-laser flow cytometer (BD)

Procedure

Culture of tumor cells
1. Coat the 10 mm culture plates with 3 ml of matrigel solution, gently rock to completely cover the plate and set aside for 30 min for gel polymerization.
2. Mince canine tumor samples (2-5 cc depending on the size of the tumor) with a scalpel blade into 1 mm pieces, digest with 10 ml of TrypLE Express for 15 min at 37 °C on a shaker table, pass the suspension through a 100 μm filter, wash with 20 ml NSC medium centrifuge at 500 x g for 5 min, and resuspend with 1 ml of NSC medium.
3. Add 1 ml of canine tumor cell suspension and 6 ml of NSC medium to the plate and place in a 37 °C humidified incubator with 5% O₂ and 5% CO₂.
4. Supplement the NSC media plates with 10 μl each of rh-EGF stock solution and rh-FGF basic stock solution every Monday, Wednesday and Friday, and the media should be changed at least once a week or if the medium indicator changes to a orange-yellow color due to rapid cell proliferation.
Harvest of tumor cells
1. When the tumor cells have grown to 100% confluency, the cells should be harvested.
2. Aspirate all of the remaining NSC medium, then add 3 ml of medium back to the plate.
3. Add 500 μl of Dispase to the plate and gently rock to spread over the cells to solubilize the matrigel coating and detach the cells. Prepare 1 ml aliquots of the Dispase to prevent excessive freeze-thaw cycles.
4. Incubate the plate at 37 °C for 20-30 min or until all cells are detached.
5. Decant the tumor cell suspension into a 15-ml conical tube and add 1x PBS to a final volume of 10 ml.
6. Centrifuge the cells at 500 x g for 5 min, decant supernatant, resuspend in 1x PBS (1 ml).
7. The cells are counted by placing 10 μl of cell suspension into an Eppendorf microcentrifuge tube and adding 10 μl of Trypan Blue.
8. Count cells using a Neubauer hemocytometer.
Serum antibody: tumor cell binding experiment
1. Place 10⁵ tumor cells into a FACS tube and add PBS to a total volume of 200 μl (produce triplicate samples).
2. Add 2 μl of serum (pre or post-treatment sera from the canine patient) into the cell suspension (1:100 dilution of serum to cells).
3. Incubate at 4 °C for 30 min.
4. After incubation, dilute cell suspension with 2 ml of FACS buffer, vortex, and centrifuge at 1,000 rpm for 5 min.
5. Decant the supernatant, repeat the wash with FACS buffer and decant.
6. Add 1 μl of the conjugated 2° antibody [goat anti-canine IgG (H&L) f(ab’)2-fluorescein isothiocyanate] and incubate at 4 °C for 30 min.
7. After incubation, add 2 ml of FACS buffer, vortex, and centrifuge at 1,000 rpm for 5 min, decant, repeat the wash and decant.
8. Resuspend the labeled cells in 200 μl FACS buffer and analyze using flow cytometer.
Observations
Use as controls tubes:
1. 2° Ab only - tumor cells + conjugated 2° antibody (no serum)
2. Unstained - tumor cells + serum (no 2° conjugated Ab)
3. Normal dog serum - tumor cells + serum from normal dog + 2° conjugated Ab

Recipes

Neural Stem Cell medium
500 ml DMEM/F12
100x 5 ml N2
50x 10 ml B27
5 ml Penicillin-streptomycin solution
Matrigel Coating solution
2.7 ml Neural Stem Cell medium
0.3 ml BD Matrigel^TM (0.3 ml aliquots in Eppendorf tubes stored at -20 °C)
rh-EGF stock solution (20 μg/ml)
20 μg recombinant human EGF
1 ml 0.1% BSA in PBS stock solution
Stored at -20 °C
rh-FGF basic stock solution (20 μg/ml)
20 μg recombinant human FGF basic
1 ml 0.1% BSA stock solution
Stored at -20 °C

Acknowledgments

This protocol was adapted from procedures in the following manuscript: Andersen et al. (2013). This work was supported by funding to B.M. Andersen from Torske Klubben Fellowship for Minnesota Residents, Medical Scientist Training Program Grant T32 GM008244, the Cancer Biology Training Grant T32 CA009138—36, and F30 CA167912; to M.A. Hunt from the American Brain Tumor Association Discovery Grant supported by the Anonymous Family Foundation; to J.R. Ohlfest from 1R21NS070955-01, R01 CA154345, R01 CA160782, the American Cancer Society grant RSG-09-189-01-LIB, Minnesota Medical Foundation, the Hedberg Family Foundation, and the Children's Cancer Research Fund.

References

Andersen, B. M., Pluhar, G. E., Seiler, C. E., Goulart, M. R., SantaCruz, K. S., Schutten, M. M., Meints, J. P., O'Sullivan, M. G., Bentley, R. T., Packer, R. A., Thomovsky, S. A., Chen, A. V., Faissler, D., Chen, W., Hunt, M. A., Olin, M. R. and Ohlfest, J. R. (2013). Vaccination for invasive canine meningioma induces in situ production of antibodies capable of antibody-dependent cell-mediated cytotoxicity. Cancer Res 73(10): 2987-2997.