Lamina propria (LP) lymphocytes, intracellular cytokine staining, and RORγt staining were performed as previously described (Ivanov et al., 2009 (link)). LP CD4+ T cells were purified by positive selection using anti-CD4 magnetic microbeads and MACS columns (Miltenyi Biotec). 3–5 × 104 CD4 T cells were co-cultured in 96-well U-bottom plates with 5 × 104 MACS purified splenic CD11c+ cells as APCs in the presence or absence of autoclaved bacterial lysates prepared from feces of SFB-monocolonized mice (SFB) or SFB-negative Jackson C57BL/6 mice (Jax) as previously described (Farkas et al., 2015 ; Goto et al., 2014 (link)). T cell proliferation was assessed 72 hours later by counting the number of live proliferated CD4 T cells.
Anti cd4 magnetic microbeads
Manufactured by Miltenyi Biotec
Anti-CD4 magnetic microbeads are a laboratory product used for the isolation and purification of CD4-positive cells from biological samples. The microbeads are coated with antibodies specific to the CD4 surface antigen, allowing for the rapid and efficient separation of CD4-expressing cells from complex cell mixtures using a magnetic field.
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3 protocols using anti cd4 magnetic microbeads
1
Isolation and Stimulation of Lamina Propria T Cells
2
Isolation of CD4+ T Cells from Lymph Nodes
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Popliteal and inguinal lymph nodes were removed from naïve, control, and EAMG animals and homogenized to single-cell suspensions. CD4+ T lymphocytes-enriched suspensions were prepared by incubation of total lymph node cells with anti-CD4 magnetic microbeads (Miltenyi Biotech) that were further separated on LS columns (Miltenyi Biotech) according to the manufacturer's recommendations. The proportions of CD4+ cells in the enriched suspensions typically ranged from 80 to 90%.
3
CD4+ T Cell Purification and Analysis
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Mononuclear cells were obtained after Ficoll-Paque PLUS (GE Healthcare Life Sciences, Piscataway, NJ) centrifugation of leukocyte-enriched fractions of whole blood. CD4 +T cells were purified by positive selection, using anti-CD4 magnetic microbeads (Miltenyi Biotech, San Diego, CA) following the manufacturers’ recommendations. The purity of the CD4 +T cells was confirmed by flow cytometric analysis using anti-human CD3-PerCP-Cyanine5.5 (clone OKT3; eBioscience, San Diego, CA) and CD4-APC (clone RPA-T4; eBioscience) antibodies. Antibodies against human CD14 and CD8 were included to confirm absence of contaminating monocytes and CD8 +T cells (anti human CD14-FITC, clone 61D3; anti-human CD8a-PE, clone HIT8a; eBioscience). Purity of CD4 +T cells was >95%. The remaining cells were predominantly CD4-low monocytes with <1% contaminating CD8 +T cells. T cells were resuspended in staining buffer (2% FBS in PBS) on ice for 30 min, washed, and then placed in IC fixation buffer (eBioscience) on ice for 10 min. Cells were washed, resuspended in staining buffer, and analyzed by flow cytometry (LSRII, BD; Franklin Lakes, NJ). Data were analyzed using FlowJo software (version 10.1 Ashland, OR).
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