Magnisort mouse t cell enrichment kit, by Thermo Fisher Scientific - Product details

1

Adoptive T Cell Transfer in Obesity

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2×10⁷ cells from either C57BL/6 WT or BSK db/db mice isolated from spleens and lymph nodes were adoptively transferred into NSG mice. Immune parameters of B6 WT or BSK db/db T cells in the recipient of NSG mice were analyzed on day 13 post-transfer. 1×10⁶ B16-F0 cells were subcutaneously inoculated in the right flank of 5–month-old male DIO and control Rag2^−/− mice. MagniSort™ Mouse T-cell Enrichment Kit (ThermoFisher) was used to enrich T cells from spleens and lymph nodes of B6 WT or BSK db/db mice. 2×10⁶ enriched T cells from either B6 WT or db/db mice were adoptively transferred into B16-F0-bearing control and DIO Rag2^−/− mice at d.p.i. 6. Immune parameters of B6 WT or db/db T cells in the recipient of B16-F0-bearing Control and DIO Rag2^−/− mice were analyzed on day 16 post-transfer.

Wang Z., Aguilar E.G., Luna J.I., Dunai C., Khuat L.T., Le C.T., Mirsoian A., Minnar C.M., Stoffel K.M., Sturgill I.R., Grossenbacher S.K., Withers S.S., Rebhun R.B., Hartigan-O’Connor D.J., Méndez-Lagares G., Tarantal A.F., Isseroff R.R., Griffith T.S., Schalper K.A., Merleev A., Saha A., Maverakis E., Kelly K., Aljumaily R., Ibrahimi S., Mukherjee S., Machiorlatti M., Vesely S.K., Longo D.L., Blazar B.R., Canter R.J., Murphy W.J, & Monjazeb A.M. (2018). Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nature medicine, 25(1), 141-151.

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Adoptive T Cell Transfer in Obesity

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2×10⁷ cells from either C57BL/6 WT or BSK db/db mice isolated from spleens and lymph nodes were adoptively transferred into NSG mice. Immune parameters of B6 WT or BSK db/db T cells in the recipient of NSG mice were analyzed on day 13 post-transfer. 1×10⁶ B16-F0 cells were subcutaneously inoculated in the right flank of 5–month-old male DIO and control Rag2^−/− mice. MagniSort™ Mouse T-cell Enrichment Kit (ThermoFisher) was used to enrich T cells from spleens and lymph nodes of B6 WT or BSK db/db mice. 2×10⁶ enriched T cells from either B6 WT or db/db mice were adoptively transferred into B16-F0-bearing control and DIO Rag2^−/− mice at d.p.i. 6. Immune parameters of B6 WT or db/db T cells in the recipient of B16-F0-bearing Control and DIO Rag2^−/− mice were analyzed on day 16 post-transfer.

Wang Z., Aguilar E.G., Luna J.I., Dunai C., Khuat L.T., Le C.T., Mirsoian A., Minnar C.M., Stoffel K.M., Sturgill I.R., Grossenbacher S.K., Withers S.S., Rebhun R.B., Hartigan-O’Connor D.J., Méndez-Lagares G., Tarantal A.F., Isseroff R.R., Griffith T.S., Schalper K.A., Merleev A., Saha A., Maverakis E., Kelly K., Aljumaily R., Ibrahimi S., Mukherjee S., Machiorlatti M., Vesely S.K., Longo D.L., Blazar B.R., Canter R.J., Murphy W.J, & Monjazeb A.M. (2018). Paradoxical effects of obesity on T cell function during tumor progression and PD-1 checkpoint blockade. Nature medicine, 25(1), 141-151.

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3

Murine T Cell Radiolabeling Protocol

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Murine T cells were obtained from the spleen of C57BL/6J mice using the MagniSort™ Mouse T cell Enrichment Kit (Invitrogen). After separation, the T cells were maintained in RPMI 1640 basal medium supplemented with 10% FBS (37 °C/5% CO₂). One day before radiolabeling, the T cells were transferred to 12 well plates with 5 × 10⁴ cells per well. Radiolabeled tracers in medium (740 KBq/1 mL) were added to each well, and the plates were incubated at 37 °C for 5 min, 10 min, 20 min, 40 min, and 60 min. After incubation, the T cells were gently collected into tubes by centrifugation, and then washed with PBS twice. The radioactivity in each tube was measured with an autogamma counter. For binding inhibition, unlabeled mPep-1-DOTA (2 mM) was added to each well. The same procedures as those in the tracer uptake assay were performed to obtain the inhibition rate at respective time points.

Hu K., Xie L., Hanyu M., Zhang Y., Li L., Ma X., Nagatsu K., Suzuki H., Wang W, & Zhang M.R. (2020). Harnessing the PD-L1 interface peptide for positron emission tomography imaging of the PD-1 immune checkpoint. RSC Chemical Biology, 1(4), 214-224.

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T Cell Isolation and Sorting

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Mouse T cells were isolated using MagniSort Mouse T cell Enrichment Kit (Invitrogen, catalog 8804-6820-74). Mouse T cells were sorted on BD FACSAria II (BD Biosciences). Human T cells were isolated using the MagniSort Human T cell Enrichment Kit (Invitrogen, catalog 8804-6810-74). Human T cells were sorted on the BD Influx Cell Sorter (BD Biosciences).

Le C.T., Vick L.V., Collins C., Dunai C., Sheng M.K., Khuat L.T., Barao I., Judge S.J., Aguilar E.G., Curti B., Dave M., Longo D.L., Blazar B.R., Canter R.J., Monjazeb A.M, & Murphy W.J. (2023). Regulation of human and mouse bystander T cell activation responses by PD-1. JCI Insight, 8(18), e173287.

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5

Tracing T Cell Proliferation

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CD4 Cre⁺Dcaf13^fl/fl (CD45.2⁺) T cells and CD4 Cre⁻Dcaf13^fl/fl (CD45.2⁺) T cells were purified from lymph nodes using a MagniSort Mouse T cell enrichment kit (no. 8804-6820-74; Invitrogen) and mixed with CD45.1⁺ T cells from wild-type mice at a ratio of 1:1. The above cells were then labeled by Celltrace Violet (C34557; Invitrogen) at 37°C for 15 min and intravenously injected into 6-Gy preirradiated CD45.1⁺ mice as hosts, and the Celltrace dilution in donor T cells was analyzed at 4 d by flow cytometry as described above.

Zhou L., Wang S., Hu W., Liu X., Xu L., Tong B., Zhang T., Xue Z., Guo Y., Zhao J., Lu L., Fan H., Qian W., Chen J., Chen W, & Wang L. (2023). T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13. The Journal of Cell Biology, 222(10), e202201096.

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6

Naive T cell activation and cytokine detection

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Naïve CD4⁺ T cells (CD4⁺CD25⁻CD62L^hiCD44^lo) and naïve CD8⁺ T cells (CD8⁺CD25⁻CD62L^hiCD44^lo) were sorted by the FACS Aria II flow cytometer, and total T cells were enriched by the MagniSort Mouse T cell Enrichment Kit (no. 8804-6820-74; Invitrogen). Sorted T cells were seeded in a 96-well or 48-well plate precoated with 2 μg/ml anti-CD3 (no. 14-0031-82; Invitrogen) and cultured in a T cell medium containing 3 μg/ml anti-CD28 (no. 16-0281-85; Invitrogen) with 30 Gy-dose irradiated antigen-presenting (APC) cells from the spleen at a ratio of 1:3. For cell cytokine detection, T cells were stimulated for 4 h at 37°C in fresh T cell medium with 50 ng/ml phorbol 12-myristate 13-acetate (PMA; no. P819; Sigma-Aldrich), 1 mg/ml ionomycin (no. I390; Sigma-Aldrich), and brefeldin A (no. 00-4506-5; eBioscience). The T cell medium was RPMI-1640 (no. 21870-076; Gibco) culture medium supplemented with 10% fetal bovine serum (no. 16000-044; Gibco), 1% penicillin and streptomycin solution (no. 516106; Sigma-Aldrich), 55 mΜ 2-mercaptoethanol (no.ES-007-E; Millipore), and 1 mM sodium pyruvate (no. 113-24-6; Sigma-Aldrich).

Zhou L., Wang S., Hu W., Liu X., Xu L., Tong B., Zhang T., Xue Z., Guo Y., Zhao J., Lu L., Fan H., Qian W., Chen J., Chen W, & Wang L. (2023). T cell proliferation requires ribosomal maturation in nucleolar condensates dependent on DCAF13. The Journal of Cell Biology, 222(10), e202201096.

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7

T Cell Activation Protocol

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Spleens were harvested from C57BL/6 (albino) mice at appx.6-8 weeks of age. T cells were negatively selected using the Magnisort mouse T cell enrichment kit (Invitrogen). The T cells were activated using 96-well flat bottom plate coated with 1mg/mL of anti-CD3 (Biolegend,17A2) antibody. The T cells were then plated using RPMI1640 medium with 10%FBS,1% PSG, 1% MEM non-essential amino acids solution, 15mM HEPES, 1mM sodium pyruvate and 55μM 2-mercaptoethanol to which soluble 1mg/mL of anti-CD28 (Biolegend, 37.51) antibody was added. The cells were then incubated for three days.

Ratnam N.M., Sonnemann H.M., Frederico S.C., Chen H., Hutchinson M.K., Dowdy T., Reid C.M., Jung J., Zhang W., Song H., Zhang M., Davis D., Larion M., Giles A.J, & Gilbert M.R. (2021). Reversing Epigenetic Gene Silencing to Overcome Immune Evasion in CNS Malignancies. Frontiers in Oncology, 11, 719091.

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8

Isolation of T Cells from Tumor-Bearing Mice

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60 tumor-bearing mice including 30 in HIFU group and 30 in sham-HIFU, and 30 naïve mice were euthanized at 14 days after HIFU procedure. Peripheral blood was immediately collected, and the spleens were respectively harvested in each group for generating single cell suspensions through a metallic mesh. Using lymphocyte density gradient centrifugation and RBC lysis buffer, the lymphocytic population was then collected, extracted and purified after removing erythrocytes and macrophages. Finally, T cells were isolated by immunomagnetic beads (MagniSort™ Mouse T cell Enrichment Kit, Invitrogen, Carlsbad, CA) in each group, according to the manufacturer’s instruction.

Ran L.F., Xie X.P., Xia J.Z., Xie F.L., Fan Y.M, & Wu F. (2023). T-lymphocytes from focused ultrasound ablation subsequently mediate cellular antitumor immunity after adoptive cell transfer immunotherapy. Frontiers in Immunology, 14, 1155229.

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Murine T-cell Cytokine Profiling

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Spleens taken aseptically from euthanized mice (4 mice/group) were dissociated individually. The splenic cell suspensions were depleted of red blood cells (RBC) using RBC lysis buffer (Sigma) and splenocytes were washed extensively with cold PBS. T cells were purified from splenocytes by MagniSort™ Mouse T-cell Enrichment Kit according to instruction (eBiosciences, CA). Purity was routinely more than 90%, as assessed by flow cytometry using monoclonal antibodies specific for mouse CD4, CD8, and CD3 cells. Cells resuspended in RPMI 1640+GlutamaxTM (Gibco) supplemented with 5% fetal bovine serum and 100 μg/ml penicillin/streptomycin were seeded in 96 well plates (1×10⁶/well) and stimulated with either YpL (4 μg/ml), the F1 antigen (4 μg/ml) or PMA (20 ng/ml) plus ionomycin (1μg/ml) as a positive control. After 72 h, the supernatants of cell cultures were collected for cytokine analysis using multiplex assay (Bio-Rad). The cells stained with CD4-phycoerythrin (PE) (clone RM4-5), CD8-allophycocyanin (APC) (clone 53-6.7) and then intracelullarly stained with IFN-γ-fluorescein isothiocyanate (FITC) (clone XMG1.2) were analyzed by Flow cytometry as mentioned in a previous report [35 (link)]. Data was collected on a Beckman Coulter FC500 and analyzed using FCS Express 4 software.

Sun W., Sanapala S., Rahav H, & Curtiss R. (2015). Oral administration of a recombinant attenuated Yersinia pseudotuberculosis strain elicits protective immunity against plague. Vaccine, 33(48), 6727-6735.

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Myeloid Cell-Mediated T Cell Suppression Assay

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For ex vivo and in vitro T cell suppression assays, T cells were purified from spleens of naive C57BL/6 J mice using the MagniSort™ Mouse T cell Enrichment Kit (eBioscience; 8802-6820), labeled with 5 µM carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher; C34570) and pre-activated prior to myeloid cell co-culture with plate-bound 0.1 µg ml⁻¹ anti-CD3 (145-2C11; eBioscience;) and 1 µg ml⁻¹ anti-CD28 (37.51; Biolegend) at 37 °C, 5% CO₂ for 16–18 h. Ex vivo T cell suppression assay with tumor-associated myeloid cells was adapted from De Henau et al.^{18 (link)}. In brief, Gl261-associated myeloid cells were isolated of from ICB R, ICB NR, and C mice. To this end, single-cell suspensions of tumor-bearing hemispheres were subjected to myelin removal (Myelin removal beads II; Miltenyi Biotec; 130-096) and CD11b⁺ cells were purified by MACS using the MagniSort™ Mouse CD11b Positive Selection Kit (eBioscience; 8802-6860). Gl261-associated CD11b⁺ cells were co-cultured with pre-activated T cells at a ratio of 1:1 (2.5 × 10⁴ CD3⁺T cells and 2.5 × 10⁴ CD11b⁺ myeloid cells) in murine T cell proliferation medium at 37 °C, 5% CO₂ for 72 h. T cell proliferation was examined by CFSE mean fluorescence intensity of living CD3⁺ CD8⁺ and living CD3⁺ CD4⁺ T cells, and percentage of cells per cell division.

Aslan K., Turco V., Blobner J., Sonner J.K., Liuzzi A.R., Núñez N.G., De Feo D., Kickingereder P., Fischer M., Green E., Sadik A., Friedrich M., Sanghvi K., Kilian M., Cichon F., Wolf L., Jähne K., von Landenberg A., Bunse L., Sahm F., Schrimpf D., Meyer J., Alexander A., Brugnara G., Röth R., Pfleiderer K., Niesler B., von Deimling A., Opitz C., Breckwoldt M.O., Heiland S., Bendszus M., Wick W., Becher B, & Platten M. (2020). Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas. Nature Communications, 11, 931.

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Magnisort mouse t cell enrichment kit

Lab products found in correlation

11 protocols using magnisort mouse t cell enrichment kit

Adoptive T Cell Transfer in Obesity

Adoptive T Cell Transfer in Obesity

Murine T Cell Radiolabeling Protocol

T Cell Isolation and Sorting

Tracing T Cell Proliferation

Naive T cell activation and cytokine detection

T Cell Activation Protocol

Isolation of T Cells from Tumor-Bearing Mice

Murine T-cell Cytokine Profiling

Myeloid Cell-Mediated T Cell Suppression Assay

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