Magnisort mouse cd4 t cell enrichment kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MagniSort™ Mouse CD4+ T cell Enrichment Kit is a laboratory tool designed for the isolation and enrichment of CD4+ T cells from mouse splenocyte or lymph node samples. The kit utilizes magnetic beads coated with antibodies specific to mouse CD4 surface antigen to selectively bind and isolate the CD4+ T cell population.

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26 protocols using magnisort mouse cd4 t cell enrichment kit

Adoptive Transfer of Allogeneic CD4+ T Cells

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CD4⁺ T cells were enriched from the spleens of TCR75.Thy1.1 mice using MagniSort Mouse CD4⁺ T cell enrichment kit (Thermo Fisher Scientific) according to manufacturer’s guidelines. Enriched CD4⁺ T cells were labeled with 5μM of CFDA (Life Technologies, Carlsbad, CA) and 3×10⁶ cells were adoptively transferred via lateral tail vein injections into B6 recipient mice. On the next day, recipient mice were injected with 5×10⁶ donor leukocytes (by tail vein) prepared as described above. At 3 days post leukocyte infusion, spleens were harvested and TCR75 T cells identified using antibodies against CD4 and Thy1.1. Proliferation was assessed via CFSE dilution on an LSR II flow cytometer (BD Bioscience).

Hudson K.E., Wong A.S., Richards A.L., Kapp L.M, & Zimring J.C. (2019). Alloimmunogenicity of an Isolated MHC Allele is Affected by the Context of MHC Mismatch in a Murine Model. Transfusion, 59(2), 744-753.

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Evaluating SOCS3 Impact on T Cell Activation

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To establish an MLR, CD4 + T cells were first obtained from the spleen and lymph nodes of a native C57/B6 mouse. BMDCs were isolated and cultured as described above. SOCS3^+/+ and SOCS3^−/− BMDCs were pretreated for 20 min at 37 °C in culture medium containing 200 μg/ml mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan). Murine CD4 + T cells were separated by negative isolation using a MagniSort™ Mouse CD4 T Cell Enrichment Kit (Thermo Fisher). Prior to culture, the purified CD4 + T cells were labeled with CFSE (Invitrogen, Ltd., UK) for subsequent assessment of T cell proliferation. Mitomycin C-treated SOCS3^+/+ or SOCS3^−/− DCs were cocultured with CD4 + T cells at a ratio of 1:4 in a 37 °C, 5% CO2 atmosphere for 5 days. T cell proliferation was assessed by evaluating CFSE dilution on the last day of culture. After stimulation with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml, Sigma) and ionomycin (5 μmol/L, Sigma) overnight, the supernatants of the cocultured cells were collected, and IL-4 and IFN-γ production was determined by ELISA.

Xu K., Wu N., Min Z., Li Z., Zhu T., Liu C., Zeng Y., Song J., Mao R., Ji H., Jiang Z, & Chen Z. (2020). Adoptive transfer of bone marrow-derived dendritic cells (BMDCs) alleviates OVA-induced allergic airway inflammation in asthmatic mice. Scientific Reports, 10, 13915.

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Purification and Characterization of Regulatory T Cells

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Rosa26-LSL-dCas9-p300 or Rosa26-LSL-dCas9-KRAB mice were backcrossed with a B6-Foxp3-eGFP mouse line (Jax stock# 006772)^{75 (link), 76 (link)} for six generations and then crossed with the B6-CD4:Cre mouse line (Jax stock# 022071)^{77 (link)} to generate heterozygous Cd4:Cre+/dCas9-KRAB+/Foxp3-eGFP+ or Cd4:Cre+/dCas9-p300+/Foxp3-eGFP+ experimental mice. Spleens and lymph nodes were harvested, dissociated, and processed with ammonium chloride potassium (ACK) lysis buffer and passed through the Magnisort mouse CD4 T cell enrichment kit (Thermo, Cat#8804-6821-74). Lymphocytes were surface-stained with antibodies for the purification of nT cells (CD4⁺CD25⁻CD62L^hiCD44^lo) through FACS using either a Beckman Culture Astiros (CD4-FITC 1:400; CD25-eFluor450 1:300; CD62L-APC 1:500; CD44-PE 1:500, and fixable viability dye e780 1:1000) or SONY SH800 (CD4-PECy7 1:500; CD25-PE 1:500; CD44-FITC 1:400; CD62L-PEcy5 1:500; Live/Dead Red 1:1000) instrument to achieve ≥98% purity. For ChIP-seq, Tn cells were purified using Magnisort mouse Naïve CD4+ Tcell enrichment kit (Thermo, Cat#8804-6824-74) rather than sorting in order to increase starting material. Antibody information: CD4 (eBiosciences, Cat#25-0042-82), CD25 (eBiosciences, Cat#12-0251-83), CD44 (eBiosciences, Cat#11-0441-85), CD62L (eBiosciences, Cat#15-0621-82).

Gemberling M.P., Siklenka K., Rodriguez E., Tonn-Eisinger K.R., Barrera A., Liu F., Kantor A., Li L., Cigliola V., Hazlett M.F., Williams C.A., Bartelt L.C., Madigan V.J., Bodle J.C., Daniels H., Rouse D.C., Hilton I.B., Asokan A., Ciofani M., Poss K.D., Reddy T.E., West A.E, & Gersbach C.A. (2021). Transgenic mice for in vivo epigenome editing with CRISPR-based systems. Nature methods, 18(8), 965-974.

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Tracking Alloreactive T Cell Responses

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Splenocytes were harvested from HODxOTII F1 and HODxOTII.Rag2p-GFP mice and processed into single cell suspensions. CD4⁺ T cells were enriched using the MagniSort Mouse CD4 T cell Enrichment Kit (ThermoFisher). HODxOTII F1. Recipient B6.Thy1.1 mice were adoptively transferred with 10×10⁶ total cells of a 1:1 mix of CellTrace-Far Red labeled HOD⁻OTII⁺ T cells and CellTrace-CFSE labeled HOD+OTII+ T cells followed the next day by a HOD RBC transfusion (100μL packed RBCs at 20% hematocrit). CellTrace dilution was assessed in CD4⁺Va2⁺Vb5⁺Thy1.2⁺ OTII T cells 3 days post-transfusion. HODxOTII.Rag2p-GFP. Enriched CD4⁺ T cells were stained with antibodies against surface antigens and CD8⁻Thy1.2⁺CD62L^hiCD44^lo T cells were sorted based on GFP expression (GFP^hi, GFPi^nt, GFP^lo) with a BD FACSAria[23 ]. At least 150,000 T cells were adoptively transferred into B6.Thy1.1 animals, followed the next day by a transfusion with 100μL of packed HOD RBCs. Splenocytes were harvested 3 days post-transfusion and CD4⁺Va2⁺Vb5⁺Thy1.2⁺ OTII T cells were evaluated for intracellular expression of Ki-67 (FoxP3 transcription factor staining buffer set, ThermoFisher), per the manufacturer’s instructions. Flow cytometric analysis was performed on a BD LSRII and data were analyzed with FlowJo (TreeStart).

Wong A.S., Gruber D.R., Richards A.L., Sheldon K., Qiu A., Hay A, & Hudson K.E. (2020). Tolerization of recent thymic emigrants is required to prevent RBC-specific autoimmunity. Journal of autoimmunity, 114, 102489.

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Enrichment and Culture of CD4+ T Cells

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Cell suspensions were prepared as described above under sterile conditions and were enriched for CD4⁺ T cells using the MagniSort Mouse CD4 T cell Enrichment Kit (no. 8804-6821-74, Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells were cultured at a concentration of 2 × 10⁶ cells per well in complete RPMI medium containing 200 nM 4-OH-tamoxifen (no. SML1666, Sigma) and 2 ng ml⁻¹ recombinant murine IL-7 (no. 217-17, Peprotech) for 48 h at 37 °C, 5% CO₂. Following incubation, cells were washed with prewarmed RPMI medium and used for adoptive transfer.

Silva-Cayetano A., Fra-Bido S., Robert P.A., Innocentin S., Burton A.R., Watson E.M., Lee J.L., Webb L.M., Foster W.S., McKenzie R.C., Bignon A., Vanderleyden I., Alterauge D., Lemos J.P., Carr E.J., Hill D.L., Cinti I., Balabanian K., Baumjohann D., Espeli M., Meyer-Hermann M., Denton A.E, & Linterman M.A. (2023). Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging. Nature Immunology, 24(7), 1124-1137.

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Purification and Characterization of Regulatory T Cells

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Foxp3+ Regulatory T Cell Conversion

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For the Foxp3⁺ regulatory T (Treg) cell conversion assay, the CD25⁺ population in harvested splenocytes from naive mice was primarily removed with the CD25 MicroBead Kit (Miltenyi Biotec), and CD4⁺ T cells were subsequently isolated with the MagniSort™ Mouse CD4 T cell Enrichment Kit (Thermo Fisher). The T cells were then labeled with 5 μM CellTrace™ Violet (Thermo Fisher) and cocultured for 4 days with either BMDCs or SPDCs at a ratio of 5:3 (50,000 T cells:30,000 DCs) with or without 2 ng/ml human TGF-β (R&D Systems) in the presence of 50 U of human IL-2 (PeproTech).

Yoo S., Jeong Y.H., Choi H.H., Chae S., Hwang D., Shin S.J, & Ha S.J. (2023). Chronic LCMV infection regulates the effector T cell response by inducing the generation of less immunogenic dendritic cells. Experimental & Molecular Medicine, 55(5), 999-1012.

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Tracking Activated T Cell Migration

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LNs and spleen were isolated from Thy1.1 congenic C57BL/6 mice, CD4⁺ T cells were isolated by magnetic sorting (MagniSort Mouse CD4 T Cell Enrichment Kit, affymetrix/eBioscience, San Diego, CA, USA), and divided into two populations, one of which was incubated with EtOH (1:1,000, 2 h) and labeled with eFluor 670 (affymetrix/eBioscience, San Diego, CA, USA; 5 µM, 10 min), and the other treated with ES048 (2 h, 1.5 µM) and subsequently CFSE labeled (affymetrix/eBioscience, San Diego, CA, USA; 5 µM, 10 min). To exclude an impact of the dye, labels were switched for half of the transfers. T cells were mixed at a 1:1 ratio and a total of 1 × 10⁷ cells of the mixture was injected into the tail vain of C57BL/6 mice. After the time periods indicated, T cells were harvested from the blood, LN, and spleen and the frequency of Thy1.1⁺/dye positive cells was analyzed by flow cytometry.

Collenburg L., Beyersdorf N., Wiese T., Arenz C., Saied E.M., Becker-Flegler K.A., Schneider-Schaulies S, & Avota E. (2017). The Activity of the Neutral Sphingomyelinase Is Important in T Cell Recruitment and Directional Migration. Frontiers in Immunology, 8, 1007.

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Investigating TGF-β1 Signaling in CD4+ T Cells

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CD4⁺ T cells from hCD4 transgenic mice were purified using a Magnisort Mouse CD4 T Cell Enrichment Kit (Affymetrix), and incubated with 10 ng/mL, 50 ng/mL, 100 ng/mL or 500 ng/mL 4T-Trap for 10 min. Cells were washed, cultured with 10 ng/mL recombinant human TGF-β1 for 1 hr, and collected into a cell lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Triton-X-100, 2mM EGTA, 10 mM NaF, 1mM Na₃VO₄ and 2 mM DTT) supplemented with protease inhibitors. Protein extracts were made, separated by SDS-PAGE gel and blotted with SMAD2/3 (D7G7) and phospho-SMAD2(Ser465/467)/SMAD3(S423/S425) (D27F4) antibodies from Cell Signaling Technology.

Li S., Liu M., Do M.H., Chou C., Stamatiades E.G., Nixon B.G., Shi W., Zhang X., Li P., Gao S., Capistrano K.J., Xu H., Cheung N.K, & Li M.O. (2020). Cancer Immunotherapy via Targeted TGF-β Signaling Blockade in TH Cells. Nature, 587(7832), 121-125.

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Naïve CD4+ T Cell Activation Signaling

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CD4⁺ T cells for T-cell activation were extracted from the spleens of experimental and control mice. We used a Magnisort^TM Mouse CD4 Naïve T cell Enrichment Kit (Invitrogen, Waltham, MA, USA) to isolate purified naïve T cells. The purity of the enriched CD3⁺CD4⁺CD44^lo T cells was 90–95%. Naïve CD4⁺ T cells were stimulated with 1 μg/mL anti-CD3 (Invitrogen, Waltham, MA, USA) and 1 μg/mL anti-CD28 antibodies (BioLegend, San Diego, CA, USA). The phosphorylation of AKT1, mTOR and RPS6KB1 was assessed by flow cytometry. For TSG101-deficient CD4⁺ T cell analysis, whole CD4⁺ T cells from the spleens of experimental or control mice were enriched using a Magnisort™ Mouse CD4 T cell Enrichment Kit (Invitrogen, Waltham, MA, USA) and stimulated with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 antibodies for 8 h. Activated CD4⁺ T cells were harvested and analyzed by flow cytometry. The antibodies used for flow cytometry are listed in Supplementary Table 1.

Jeong J., Kang I., Kim Y., Ku K.B., Park J.H., Kim H.J., Kim C.W., La J., Jung H.E., Kim H.C., Choi Y.J., Kim J., Kim J, & Lee H.K. (2023). Regulation of c-SMAC formation and AKT-mTOR signaling by the TSG101-IFT20 axis in CD4+ T cells. Cellular and Molecular Immunology, 20(5), 525-539.

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