Ack rbc lysis buffer

A medulloblastoma tissue sample was obtained after appropriate patient consent at the Lucille Packard Children’s Hospital (Stanford, CA), in accordance with Institutional Review Board protocols (Protocol ID #: 12625; IRB #: 4593 (Panel: 5)). Pathology of the tumor was confirmed upon histopathologic analysis by institutional neuropathologists prior to further analysis.
The tumor sample was mechanically dissociated in a solution containing Hank’s Balanced Salt Solution (HBSS), nonessential amino acids, sodium pyruvate, sodium bicarbonate, HEPES, Glutamax-1, antibiotic-antimycotic, DNase, and collagenase IV. All media components were from Cellgro, except for DNase and collagenase IV, which were from Worthington. The suspension was washed two times with HBSS, filtered through a 70-μm strainer and resuspended in a 0.9 M sucrose solution in HBSS without Ca²⁺/Mg²⁺ (Cellgro) to remove debris and dead cells. The cells were treated with ACK/RBC lysis buffer (Gibco), then washed twice in phosphate buffered saline (PBS). Cells were plated in tumor stem media (TSM) consisting of Neurobasal-A medium (Invitrogen), B27-A (Invitrogen), human bFGF (20 ng/ml) (Shenandoah Biotech), human EGF (20 ng/ml) (Shenandoah Biotech), human recombinant LIF (20 ng/ml) (Millipore), and heparin (10 ng/ml).

Mouse spleens were collected in 50 mL Falcon tubes decked with 70 μm nylon cell strainers and kept in ice. Spleens were gently passed through the cell strainer with the help of syringe plungers. Cells were then centrifuged at 700 g for 5 min at 4 °C. Cell pellets were resuspended with 1 ml Gibco™ ACK RBC Lysis buffer per spleen and incubated for 5 min at room temperature with occasional shaking. The reaction was then stopped by adding 30 ml of RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin and 50μM beta-mercaptoethanol. After centrifuging at 700 g for 5 min at 4 °C, the cell pellets were resuspended in 10 ml of ice-cold RPMI 1640 media supplemented with 10% FBS, 1% penicillin-streptomycin and 50μM beta-mercaptoethanol, and plated at density of 250,000 cell/well in the 96-well plates provided with the ELISPOT assay kit R&D Systems, #EL485, Minneapolis, MN. Customized E6 or E7 peptide libraries (GenScript) were added to the wells at the final concentration of 5 μg/ml, unless otherwise indicated. After an overnight incubation at 37°C, plates were processed as per manufacturer (R&D Systems, #EL485)’s instructions. All microplates were read under the CTL Immunospot Analyzer (ImmunoSpot^®).