Accumax cell dissociation solution

For flow cytometry analysis, cells were detached from the tissue culture plate by incubating in 150μl of Accumax Cell Dissociation Solution (Innovative Cell Technologies, Inc.) for 5 min at 37°C, transferred to V-bottom 96-well plates, pelleted by centrifugation at 800 x g for 5 min, resuspended in 1% paraformaldehyde (PFA) and incubated at 4°C for at least 30 min. Fixed cells were then pelleted at 800 x g for 5 min and resuspended in 150μl of 1×PBS containing 3% FBS. Plates were stored at 4°C if flow cytometry was not carried out immediately. Samples were analyzed using a Stratedigm S1000 flow cytometer equipped with 405nm, 488nm and 561nm lasers. Data was analyzed using FlowJo Software (Treestar).

Mice: All animal studies were conducted with institutional oversight under an IACUC-approved animal care and use protocol (ACUP) at Certis Oncology Solutions using adult female NOG mice (Taconic, Germantown, NY, USA).
Preparation of Cells for Xenotransplantation:
Cells were grown and subcultured in GBM media as described previously [28 (link)]. On the day of implantation, tumor spheres were dissociated into a single cell suspension using Accumax Cell Dissociation Solution (Innovative Cell Technologies, Novi, MI, USA. #AM105) according to standard manufacturer recommendations. Cell numbers and viability were quantified using the Countess II Automated Cell Counter (Invitrogen, Waltham, MA, USA). Cells were resuspended in either GBM media (intracranial implants) or PBS:Matrigel (50:50) (subcutaneous implants) (Corning, Tewksbury, MA, USA. #356237) at the appropriate density for in vivo implantation. Cell suspensions were kept on ice until injection.

B cells were isolated from bone marrow (BM), subchondral BM (SBM) and synovium of mice. BM cells were flushed out from the femora and tibiae. To harvest SBM cells, all the joints including knees, fore and hind paws were separated, and muscles were removed. Subchondral (between the articular cartilage and growth plate) and synovial tissues were cut into small pieces and digested using Accumax Cell Dissociation Solution (Innovative Cell Technologies) at room temperature for one hour to yield SBM cells. Both SBM and synovial B cells were isolated together given their anatomic proximity and are referred to throughout the manuscript as SBM (SBM plus synovial B cells). RBCs from BM and SBM cells were eliminated using RBC lysis buffer. B cells were then purified, based on CD19 expression using microbeads conjugated with anti-CD19 antibody (Ab) and magnetic isolation (Miltenyi Biotec, Auburn, CA)^{50 (link)}. Human peripheral blood mononuclear cells (PBMCs) were purified from heparinized PB by Ficoll-Hypaque density gradient centrifugation (Pharmacia Biotech), and B cells were isolated based on CD19 expression using CD19 microbeads and magnetic isolation (Miltenyi Biotec, Auburn, CA)^{4 (link)}.

Cell cycle effects were analyzed after 24 h (data not shown) and 48 h treatment using propidium iodide staining to label DNA content. Briefly, cells were detached with Accumax cell dissociation solution (Innovative Cell Technologies, San Diego, CA USA), collected in tubes with 1% fetal bovine serum (FBS) in Phosphate Buffered Saline (PBS) and centrifuged for 5 m at 300 rcf at 4 °C. The cell pellet was washed twice with 1% FBS in PBS, filtered, and resuspended in 70% cold ethanol for overnight fixation. The following day, samples were centrifuged for 10 m at 500 rcf at 4 °C, resuspended in cold PBS. Samples were centrifuged again for 5 m at 300 rcf at 4 °C and resuspended in DNA staining solution [2% propidium iodide (Sigma Aldrich), 0.1% Triton X-100 (Sigma Aldrich), in PBS]. Samples were then incubated for 30 m at room temperature and analyzed with an excitation of wavelength of 488 nm and emission of 617 nm. Only C2 and D17 cell lines were analyzed due to the presence of frequent doublets with CMT-12 cells resulting in an artificial accumulation in the G2/M phase.

Passage 2 MSCs isolated previously from horses of ELA haplotype ELA-A2 and identified as either MHC class II-negative or class II-positive were thawed from liquid nitrogen storage and expanded by in vitro culture as described previously until 30 × 10⁶ to 50 × 10⁶ cells were attained (Table 1) [24 (link)]. This number of cells was chosen based on a clinically relevant range as well as previous studies in the literature from other species in which the stem cell dose was calculated according to body weight [25 (link)-27 (link)]. MSCs were lifted from tissue culture plates using Accumax cell dissociation solution (Innovative Cell Technologies Inc., San Diego, CA, USA), washed three times with phosphate-buffered saline to remove residual fetal bovine serum (FBS), counted, and resuspended in 1 ml phosphate-buffered saline for injection. MSCs were maintained at room/ambient temperature during transport from the laboratory to the recipient horse [35 (link)]. Immediately prior to MSC injection, an approximately 10 cm × 10 cm area on the left side of the recipient’s neck was clipped and prepared in a sterile manner. The MSC suspension was then injected intradermally using a 20 G needle. Recipient horses were monitored for increased respiratory rate, heart rate, and body temperature as well as swelling, heat, and pain at the site of injection for the duration of the study.