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Dynabeads untouched mouse cd4 cells kit

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The Dynabeads Untouched Mouse CD4 Cells Kit is a magnetic bead-based system designed for the isolation of untouched mouse CD4+ T cells from a cell suspension. The kit utilizes a negative selection approach to enrich the target cell population without directly labeling the cells of interest.

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Lab products found in correlation

Ack lysing buffer, by Thermo Fisher Scientific (4 mentions) Ack buffer, by Lonza (3 mentions) Facsaria 2 cell sorter, by BD (3 mentions) Ack lysis buffer, by Thermo Fisher Scientific (2 mentions) Dispase in hbss, by Thermo Fisher Scientific (2 mentions) Collagenase 2, by Thermo Fisher Scientific (2 mentions) Rpmi 1640, by Merck Group (2 mentions) Noradrenaline, by Merck Group (2 mentions) Facsaria 2, by BD (2 mentions) Roswell park memorial institute (rpmi), by BD (2 mentions) Fetal bovine serum (fbs), by Gemini Bio (2 mentions) Facsaria, by BD (2 mentions) Cd8 t cell isolation kit, by STEMCELL (2 mentions) Easysep mouse cd4, by STEMCELL (2 mentions) Dynabeads untouched mouse cd8 cells kit, by Thermo Fisher Scientific (2 mentions) Rag12m, by Taconic Biosciences (2 mentions) Rag1tm1mom, by Jackson ImmunoResearch (2 mentions) Cd43 microbead, by Miltenyi Biotec (2 mentions) Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions) Anti nk1.1 clone pk136, by BioXCell (1 mentions)

48 protocols using dynabeads untouched mouse cd4 cells kit

1

Isolation and Transfer of ILC2 Precursors

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Bones were cleaned, washed in 70% EtOH and subsequently in PBS and crushed using a pestle. Bones were rinsed with PBS and bone marrow was resuspended in PBS and filtered through a 70 μM cell strainer. Red blood cells were lysed using ACK buffer (Lonza) and lineage-positive cells were depleted using the Dynabeads untouched mouse CD4 cells kit according to the manufacture’s protocol (Thermo Fisher Scientific). Remaining cells were stained and ILC2ps were sort-purified as Lin− Sca-1high CD127+ CD25+ cells 14 (link). 1×104 sort-purified ILC2ps were injected i.v. and mice were used at least 4 weeks after adoptive transfer.
Klose C.S., Mahlakõiv T., Moeller J.B., Rankin L.C., Flamar A.L., Kabata H., Monticelli L.A., Moriyama S., Putzel G.G., Rakhilin N., Shen X., Kostenis E., König G.M., Senda T., Carpenter D., Farber D.L, & Artis D. (2017). The neuropeptide Neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature, 549(7671), 282-286.
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2

Bone Cell Isolation and Purification

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Femurs, tibiae, hips and spines were dissected and cleaned from surrounding tissue. For all cell sorting and flow cytometry analyses, bones were crushed in cell suspension medium (RPMI 1640 (Sigma) containing 2% fetal bovine serum) using a mortar and a pestle. Dissociated cells were filtered through a 40 μm filter, spun down at 1500 rpm for 5 min, then incubated with 5 ml ACK lysis buffer (Thermo Fisher Scientific) for 5 min at room temperature for red blood cell lysis. Neutralization was achieved with 20 ml cell suspension medium. Cells were then lineage-depleted using Dynabeads Untouched Mouse CD4 Cells Kit (Thermo Fisher Scientific) according to manufacturer’s recommendations, using a home-made lineage cocktail.
For cell extraction from bones, crushed bone chips were washed four times each with 10 ml of cell suspension medium, then incubated with 10 ml of digestion medium (1 mg/ml of each Collagenase II and Dispase in HBSS, all from Gibco) for 30 min at 37 °C in a water bath. Cell suspension was then removed and filtered through a 40 μm filter and the digestion reaction was stopped by adding 40 ml of cell suspension medium. From this point on, cells were treated exactly the same as bone marrow cells above.
Baccin C., Al-Sabah J., Velten L., Helbling P.M., Grünschläger F., Hernández-Malmierca P., Nombela-Arrieta C., Steinmetz L.M., Trumpp A, & Haas S. (2019). Combined single-cell and spatial transcriptomics reveals the molecular, cellular and spatial bone marrow niche organization. Nature cell biology, 22(1), 38-48.
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3

T cell activation and co-culture with MSCs

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CD4+ cells were isolated from suspensions of splenocytes using Dynabeads Untouched Mouse CD4 Cells Kit (Thermo) according to manufacturer’s protocol. Cells were counted and plated in RPMI/10% FCS at density 107 cells/ml to 24 well plate covered with anti-mouse CD3 monoclonal antibodies (clone 37.81, Biolegend). Cells were collected after 48 hours of incubation (37°C, 5%CO2), counted and plated in 24 well plate with MSC at MSC:T cell ratio 1:10 for 48 hours (37°C, 5%CO2). T cells were collected, counted and stained with fluorophore-conjugated monoclonal antibodies to evaluate surface markers as mentioned above. Stained cells were analysed by flow cytometry.
Nenasheva T., Nikolaev A., Diykanov D., Sukhanova A., Tcyganov E., Panteleev A., Bocharova I., Serdyuk Y., Nezlin L., Radaeva T., Adrianov N., Rubtsov Y, & Lyadova I. (2017). The introduction of mesenchymal stromal cells induces different immunological responses in the lungs of healthy and M. tuberculosis infected mice. PLoS ONE, 12(6), e0178983.
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4

Adoptive Transfer of Activated CD4+ T Cells

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CD4+ T cells were isolated from the spleen using the Dynabeads® Untouched™ Mouse CD4 Cells Kit (Thermo Fisher Scientific Inc.) after splenocytes were obtained from unstressed WT C57BL/6 mice (the details of isolating splenocytes are described above). Isolated CD4+ T cells were treated with noradrenaline (Sigma-Aldrich, 10 μM) for 30 minutes at 37° C. After washing the cells with PBS, CD4+ T cells were re-suspended in PBS and 1 × 105 cells (200 μl) were injected into recipient mice (naïve WT C57BL/6 mice) via tail vein 24 hours prior to IR.
Abe C., Inoue T., Inglis M.A., Viar K.E., Huang L., Ye H., Rosin D.L., Stornetta R.L., Okusa M.D, & Guyenet P.G. (2017). C1 neurons mediate a stress-induced anti-inflammatory reflex in mice. Nature neuroscience, 20(5), 700-707.
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5

Isolation and Transfer of ILC2 Precursors

Cited 1 time
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Bones were cleaned, washed in 70% EtOH and subsequently in PBS and crushed using a pestle. Bones were rinsed with PBS and bone marrow was resuspended in PBS and filtered through a 70 μM cell strainer. Red blood cells were lysed using ACK buffer (Lonza) and lineage-positive cells were depleted using the Dynabeads untouched mouse CD4 cells kit according to the manufacture’s protocol (Thermo Fisher Scientific). Remaining cells were stained and ILC2ps were sort-purified as Lin− Sca-1high CD127+ CD25+ cells 14 (link). 1×104 sort-purified ILC2ps were injected i.v. and mice were used at least 4 weeks after adoptive transfer.
Klose C.S., Mahlakõiv T., Moeller J.B., Rankin L.C., Flamar A.L., Kabata H., Monticelli L.A., Moriyama S., Putzel G.G., Rakhilin N., Shen X., Kostenis E., König G.M., Senda T., Carpenter D., Farber D.L, & Artis D. (2017). The neuropeptide Neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature, 549(7671), 282-286.
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6

Isolation and Activation of Murine CD4+ T Cells

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CD4+ T lymphocytes were isolated from spleens of 4-6 weeks old Balb/c mice. Briefly, spleens were mechanically mashed through a 70 μm cell strainer following erythrocyte lysis using the ammonium-chloride-potassium buffer, and the obtained cell suspension was filtered through a 40 μm cell strainer. CD4+ T cells were isolated using Dynabeads Untouched Mouse CD4 Cells Kit (ThermoFisher, USA) according to the manufacturer's instructions. Isolated CD4+ T cells were cultured in RPMI 1640 supplemented with 10% FBS. The medium used in T cell cultures was depleted from exosomes by 1-hour centrifugation at 100 000 g. T cell activation was performed with Mouse T-Activator CD3/CD28 Dynabeads (ThermoFisher, USA). After 3 days of culture (density about 4 × 106 T cells/ml), the medium was collected for exosome isolation or used as conditional medium.
Rolski F., Czepiel M., Tkacz K., Fryt K., Siedlar M., Kania G, & Błyszczuk P. (2022). T Lymphocyte-Derived Exosomes Transport MEK1/2 and ERK1/2 and Induce NOX4-Dependent Oxidative Stress in Cardiac Microvascular Endothelial Cells. Oxidative Medicine and Cellular Longevity, 2022, 2457687.
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7

Treg Cell-Mediated Suppression of CD4+ T Cell Proliferation

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The responder CD4+ T cells were isolated from CD45.1+ congenic mice by using the Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Thermo Fisher Scientific), and then labeled with CTV. Dynabeads™ FlowComp™ Mouse CD4+CD25+ Treg Cells Kit (Thermo Fisher Scientific) was used to isolate Treg cells from the spleens of naïve B6 or Irf4fl/flCd4-Cre mice, or from the spleens of Irf4fl/flCd4-Cre recipients at day 7 after heart transplantation (treated with either Rat IgG or anti-CTLA-4 plus anti-PD-L1 mAbs). CTV-labeled CD45.1+CD4+ T cells were added at 1×105 cells/well in 96-well round bottom tissue-culture plates together with or without equal numbers of Treg cells from different groups, and stimulated with T-cell–depleted mitomycin C-treated B6 splenocytes and 1 μg/ml soluble anti-CD3e mAb. Three days later, CD45.1+CD4+ T cells were analyzed for proliferation by CTV dilution using a LSR II flow cytometer.
Wu J., Zhang H., Shi X., Xiao X., Fan Y., Minze L.J., Wang J., Ghobrial R.M., Xia J., Sciammas R., Li X.C, & Chen W. (2017). Ablation of transcription factor IRF4 promotes transplant acceptance by driving allogenic CD4+ T cell dysfunction. Immunity, 47(6), 1114-1128.e6.
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8

Adoptive Transfer of Antigen-Specific T Cells

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On day 0, CB6F1 and WT B6 mice were adoptively transferred intravenously with 1 × 106 CD45.1+ TEa cells, which were isolated from spleens of CD45.1+ TEa mice using the Dynabeads untouched mouse CD4 cells kit (Thermo Fisher Scientific). WT B6 mice were also transplanted with Balb/c tail skins on day 0, whereas CB6F1 mice were intraperitoneally injected with 500 μg anti-NK1.1 (clone PK136, Bio X Cell) on days −3 and 15 to deplete NK cells. The following experiments were then performed: (1) on different days after TEa cell transfer, TEa cell states in peripheral blood and spleens were determined by flow cytometric analysis; (2) on day 6 after TEa-cell transfer, TCR(Vα2+ Vβ6+)CD45.1+CD4+ TEa cells were sorted from splenocytes, followed by RNA-seq analysis as described later; and (3) on day 30 after TEa cell transfer, TCR(Vα2+ Vβ6+)CD45.1+CD4+ TEa cells were sorted from splenocytes and further adoptively transferred into B6.Rag1−/− mice. One day later, B6.Rag1−/− mice were transplanted with Balb/c tail skins to determine the antigraft function of TEa cells.
Zou D., Dai Y., Zhang X., Wang G., Xiao X., Jia P., Li X.C., Guo Z, & Chen W. (2020). T cell exhaustion is associated with antigen abundance and promotes transplant acceptance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(9), 2540-2550.
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9

Immunophenotyping and Cell Sorting Protocol

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Cells were stained with the Ab combinations listed in Table S2 in PBS/0.5% BSA/2 mM EDTA. Samples were acquired on a LSR II or Fortessa analyzer (BD Biosciences) and analyzed with FlowJo10 software. For intracellular staining, cells were fixed overnight (ON) at 4°C and then permeabilized and stained with the primary Ab for 1 h at room temperature and, when necessary, with a secondary Ab for 1 h on ice using the eBioscience Foxp3/Transcription Factor Staining Kit (Thermofisher). For cytokine staining, CD4+ T cells were first magnetically enriched using the Dynabeads Untouched Mouse CD4 Cells Kit (Thermofisher) and then stimulated for 2 h at 37°C with PMA and ionomycin (0.5 μg/ml each) in the presence of BD GolgiPlug (BD Biosciences) in IMDM supplemented with 10% FCS, MEM, Hepes (10 mM), GlutaMAX (Gibco), sodium pyruvate (1 mM), and 2-mercaptoethanol (50 μM). Cells were stained and analyzed as above. For sorting HSPC subsets, cells were stained for lineage marker expression (Table S2) before depletion with Dynabeads sheep anti-rat IgG beads (Thermofisher). Lineage-negative cells were stained as above and sorted in the presence of DAPI (1 μM/ml) using a FACS ARIA II or Fusion (BD Biosciences). Sort purity was >98%.
Cova G., Taroni C., Deau M.C., Cai Q., Mittelheisser V., Philipps M., Jung M., Cerciat M., Le Gras S., Thibault-Carpentier C., Jost B., Carlsson L., Thornton A.M., Shevach E.M., Kirstetter P., Kastner P, & Chan S. (2021). Helios represses megakaryocyte priming in hematopoietic stem and progenitor cells. The Journal of Experimental Medicine, 218(10), e20202317.
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10

Isolation of Mouse CD4+ T Cells

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MesLNs were collected and CD4+ T cells isolated using the Dynabeads™ Untouched™ Mouse CD4 Cells Kit (Thermo Fisher) according to the manufacturer’s instructions.
Classon C.H., Li M., Clavero A.L., Ma J., Feng X., Tibbitt C.A., Stark J.M., Cardoso R., Ringqvist E., Boon L., Villablanca E.J., Rothfuchs A.G., Eidsmo L., Coquet J.M, & Nylén S. (2021). Intestinal helminth infection transforms the CD4+ T cell composition of the skin. Mucosal Immunology, 15(2), 257-267.
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