Cell culture and establishment of a cell line stably transfected with Rae-1
The cancer cell lines CT26, TC1, B16F10, LLC, K7M3, and YAC-1 were obtained from American Type Culture Collection (Rockville, MD, USA). CT26, TC1, K7M3, B16F10, and LLC cells were grown in Dulbecco's modified Eagle’s medium (Mediatech, Inc., Manassas, VA, USA) supplemented with glutamine, heat-inactivated 10% fetal calf serum, and 10 U/ml penicillin and streptomycin. YAC-1 cells were grown in RPMI-1640 medium (Mediatech, Inc.) supplemented with heat-inactivated 10% fetal calf serum and 10 U/ml penicillin and streptomycin.
The murine gene Rae-1δ (Open Biosystems) was subcloned into a pBMN–green fluorescent protein (GFP) plasmid. Retroviruses were produced by transfecting mRae-1δ/pBMN-GFP constructs into Phoenix-ECO packaging cells. CT26 cells were infected with the retrovirus-containing supernatant derived from the transduced HEK293 cells. Cell colonies were expanded from a single cell expressing GFP. Both Rae-1δ/GFP and GFP-positive CT26 cells were confirmed using flow cytometry.
Stable transfected cells were washed twice in phosphate-buffered saline (PBS), counted, suspended in 100 μl of sterile PBS, and then transferred to a 0.5-ml tuberculin syringe. Six- to seven-week-old BALB/C mice were injected with 35 × 106 cells in a 50-μl volume in each foot. The mice received injections every 3 days for 18 days (6 injections total). On day 18, the mice were humanely killed, and B cells were isolated from lymph nodes for fusion.
Myeloma cells expansion
One week before fusion was to be performed, we began growing SP2/0-Ag14 myeloma cells in a 10-cm petri dish containing RPMI medium supplemented with 10% FBS to ensure that 1 × 108 cells would be available for fusion.
Mouse lymph nodes harvest
For the mouse lymph node harvest, we first prepared RPMI medium containing 10% FBS, 1× PN/SM and 1× hypoxanthine, aminopterin, and thymidine (HAT) medium, and we prewarmed 50% polyethylene glycol (PEG; Sigma) in a 37°C incubator. We then euthanized the mice and aseptically harvested the lymph nodes. We transferred the lymph nodes into a sterile 10-cm petri dish containing 10 ml of serum-free RPMI medium. We used forceps to manipulate the lymph nodes to release cells and transferred the lymphocyte suspension to a sterile 50-ml conical centrifuge tube that we then filled with serum-free RPMI medium. We washed the cells 2 times with serum-free RPMI medium. To harvest the Sp2/0-Ag14 myeloma cells, we transferred the cells into 50-ml conical centrifuge tubes and centrifuged them at 1150 rpm for 3 min at room temperature. After aspirating and discarding the supernatant, we resuspended the SP2/0-Ag14 cells in serum-free RPMI medium and washed them 2 times. We used a hemacytometer and staining with trypan blue to count the cells in each suspension and assess their viability.
Cell fusion for mAbs
On the day fusion was performed, mouse lymph nodes were harvested to obtain the lymphocytic cells. Lymphocytes and myeloma cells were harvested, washed, and then mixed together. Cell fusion was performed in the presence of polyethylene glycol (PEG). The resulting pellet was harvested and placed in tissue culture plates. After incubation with hypoxanthine, aminopterin, and thymidine (HAT) medium and feeding for 10 days, the hybridomas were ready for screening.
Lymphocytes and Sp2/0-Ag14 myeloma cells were mixed in a 50-ml conical tube at a ratio of 1:0.8. The tube was then filled with serum-free RPMI medium, and the cell mixture was subjected to centrifugation at 1350 rpm for 5 min at room temperature. After the supernatant was aspirate and discarded, 1 ml of sterile PEG was added to the cell pellet. The cell pellet was then agitated for 45 sec, and 40 ml of prewarmed serum-free RPMI medium was added to stop the reaction. The mixture was then subjected to centrifugation at 1150 rpm for 5 min at room temperature. The supernatant was aspirated, and the cell pellet was resuspended in HAT medium. The cells were then placed in 96-well flat-bottom plates.
Costar EIA/RIA plates (Fisher Scientific, Hampton, NH) were coated with 20 ×106 cells/plate and allowed to dry overnight before storage in a -20°C freezer until use. For ELISA, the cells were washed with PBS containing 0.05% Tween 20 (PBST) 3 times and blocked by incubation in PBST containing 2% bovine serum albumin for 1 hour at room temperature. Culture supernatant (100 μl) was then added, and the cells were incubated for 1 hour at room temperature and then washed with PBST 3 times. Goat anti-mouse immunoglobulin G (IgG) Fc, horseradish peroxidase (HRP) conjugate (100 μl; Jackson Immunoresearch: 115-035-071) was then added, and the cells were incubated at room temperature for 1 hour and washed 5 times with PBST before the substrate was added. Absorbance was read at 450 and 620 nm.
Different amounts of Rae-1β recombinant protein were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gel and transferred to nitrocellulose membranes using the iBlot gel transfer device (Invitrogen, Grand Island, NY). The membranes were blotted with anit–Rae-1 primary antibody and HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Dallas, TX) to detect the protein of interest.
Cells were gently trypsinized and washed once with serum-containing media. The cell pellets were washed with cold PBS free of Ca2+ and Mg2+ and then resuspended in 100 μl of PBS. The cells were stained with the indicated primary and secondary antibodies for 30 min at 4°C. The expression of the indicated genes was analyzed using a FACSCalibur flow cytometer (BD Biosciences).
CT26 cells (1 × 106) were transfected with 1 μg of plasmid DNA using the X-tremeGENE HP DNA transfection reagent (Roche Diagnostics, Indianapolis, IN).
Cells were seeded on coverslips in 12-well plates (1×105 cells/well). The next day, the cells were washed in 1× HEPES-buffered Hank’s balanced salt solution (HEPES/HANKS) buffer and then fixed in 1% paraformaldehyde for 30 min. After the paraformaldehyde was removed carefully and discarded, the cells were rinsed in PBS for 5 min 3 times. The cells were blocked in 1% goat serum in PBS for 1 h, the primary antibody was added, and the cells were incubated overnight at 4°C or for 2 h at room temperature if needed. After the cells were washed with PBS for 5 min 3 times, the secondary antibody was added, and the cells were incubated for 60 min room temperature. The cells were then washed with PBS for 5 min 3 times, rinsed with water, mounted on slides, and coated with anti-fade reagent (Life Technologies, Carlsbad, CA). The slides were stored in the dark before they were observed under a fluorescence microscope.
Tumor inoculation and frozen tissue section preparation
Cells stably transfected with Rae-1 (CT26–Rae-1 cells) and control cells (CT26-GFP cells) were subcutaneously injected into BALB/c mice (2 × 105 cells/mouse). Fourteen days after injection, the mice were humanely killed and their tumors harvested. The tumors were frozen in (optimum cutting temperature) OTC solution, and tissues sections were cut and mounted on glass slides.
Frozen tumor sections were sequentially fixed with cold acetone, acetone plus chloroform (1:1), and acetone. Tissue sections were blocked with blocking buffer (5% normal horse serum and 1% normal goat serum in PBS) and then incubated with the primary antibody overnight at 4°C. The next day, the tissues were incubated with the secondary antibody for 1 hour at room temperature. Nuclei were counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO).