Pancreatic tumors were dissociated using the gentleMACS Dissociator as described above to isolate tumor-infiltrating lymphocytes (TILs). To expand TILs ex vivo, CD8 T cells isolated using CD90.2 magnetic beads (Miltenyi Biotec) were incubated at a ratio of 5:1 with irradiated K562 artificial antigen presenting cells engineered to express CD32 (FcRγII) and CD137L (41BB-L) and loaded with 16 μg/mL anti-CD3 (clone 145-2c11) and 2 μg/mL anti-CD28 (clone 37.51) antibody (BD Biosciences). TIL-K562 co-cultures were incubated in RPMI full growth medium supplemented with 10% (v/v) FBS, penicillin /streptomycin, 50 μM 2-mercaptoethanol, L-glutamine, and the proliferative cytokines IL-2, (5 U/mL), IL-7 (5 ng/mL), and IL-15 (5 ng/mL) purchased from (PeproTech). After 7–10 days in culture, expanded tumor reactive TILs will were incubated at ratios of 1:5, 1:10 and 1:20 with KPC1242 for 24 h and tumor cell killing measured using IncuCyte S3 (Essen BioScience).
Cd90.2 magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
CD90.2 magnetic beads are a specialized laboratory tool used for the isolation and purification of specific cell types. These beads are coated with antibodies that recognize the CD90.2 surface marker, allowing for the selective separation of cells expressing this antigen.
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Lab products found in correlation
Live dead fixable aqua dead cell staining kit, by Thermo Fisher Scientific (3 mentions)
Cytofix cytoperm kit, by BD (3 mentions)
Ebioscience cell stimulation cocktail plus protein transport inhibitor, by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Anti cd3 clone 145 2c11, by BD (1 mentions)
Incucyte s3, by Sartorius (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Facsaria, by BD (1 mentions)
C1 inh, by CSL Behring (1 mentions)
Macs ls, by Miltenyi Biotec (1 mentions)
Catalog no 25030081, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Pan t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
9 protocols using cd90.2 magnetic beads
1
Expansion and Characterization of Pancreatic TILs
2
Adoptive transfer of T cells
3
Intracellular Cytokine Staining of Activated T Cells
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Purification of T cells or T cell subsets from spleen or resected tumors was performed using CD90.2 magnetic beads (Miltenyi) according to the manufacturer’s protocol or via cell sorting using FACSAria (BD Biosciences). For ex vivo T cell activation, 1×106 T cells were stimulated at 37°C with eBioscience Cell Stimulation Cocktail plus protein transport inhibitor (Thermo Fisher Scientific) for 6 hours. Cells were washed and stained for intracellular cytokines using BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against interferon (IFN)-γ, granzyme B, and tumor necrosis factor (TNF)-α after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, California, USA).
4
GVHD Model with Mannose-Binding Lectin Deficiency
5
Allogeneic Bone Marrow Transplant with SIRT3 Deficient T Cells
6
Retinal Ganglion Cell Isolation
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To isolate RGCs, we used a method that has previously been verified with slight modifications. Briefly, animals from each group were euthanized with CO2 asphyxiation and their retinas were harvested in cold neurobasal medium and placed in a 37°C water bath for 5 minutes. The neurobasal media was removed and replaced with fresh pre-warmed neurobasal media containing papain (0.06mg/ml or 33.4 U/mg) and 5mM L-cysteine and incubated 20 minutes at 37°C. The papain solution was removed and replaced with neurobasal containing 2mM L-Glutamine (Gibco; Catalog no. 25030081) B-27 supplement (Gibco; Catalog no. 17504044) and 10% FBS (Gibco; Catalog no. 26140079). The retinas were dissociated by gently pipetting up and down with a wide bore 1ml pipette tip. Cells were then centrifuged at 450g for 8 minutes and resuspended in 90 μl of isolation buffer (DPBS + 0.5% BSA + 2mM EDTA) containing 25 μg/ml DNAse I + 5mM MgCl2. Cells were filtered through a 30μm cell strainer and incubated with CD90.2 magnetic beads (Miltenyi Biotec; Catalog no. 130-121-278) at 4°C for 10 minutes and then isolated using MACs LS (Miltenyi Biotec; Catalog no. 130-042-401) columns following the manufacturer’s instructions. After isolation, cells were washed with DPBS, centrifuged at 450g for 8 minutes and their pellets stored at −70°C prior to proteomics sample preparation and LC/MS analysis.
7
Murine Bone Marrow Transplantation Protocol
8
Isolation and Activation of T Cells from Murine Models
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T cells from the spleen of OT-I and OT-II mice or lung tumors of treated KP mice were isolated from single-cell suspensions using CD90.2 Magnetic beads (Miltenyi) according to the manufacturer’s protocol. For ex-vivo T cell activation, 1 × 106 T cells isolated from lung tumors were stimulated at 37°C with eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (ThermoFisher Scientific) for 6 hours. Cells were washed and stained for intracellular cytokines using BD cytofix/cytoperm kit (BD Biosciences) according to the manufacturer’s instructions at the end of the 6-hour culture. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against IL-2, TNF-α, CD107a, and IFN-γ, or isotype-matched antibodies after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, CA).
9
Intracellular Cytokine Profiling of Tumor-Infiltrating T Cells
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T cells from lung tumors of treated mice were isolated from single cell suspensions using CD90.2 Magnetic beads (Miltenyi) according to the manufacturer's protocol. 1 x 10 6 isolated T cells were then stimulated at 37°C with eBioscience™ Cell Stimulation Cocktail plus protein transport inhibitors (Invitrogen) for 6 hours. Cells were washed and stained for intracellular cytokines using BD cytofix/cytoperm kit (BD Pharmingen) according to the manufacturer's instructions at the end of the 6-hour culture. Briefly, cells were first stained for surface markers, including CD4, CD8, and CD3, followed by intracellular staining with fluorophore-conjugated antibodies against IL-2, TNFα, CD107a, and IFN-γ, or isotype-matched antibodies after fixation and permeabilization. In all stained samples, dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Staining Kit (Invitrogen, Carlsbad, CA).
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