CD4+ T cells were isolated from splenocytes using anti-mouse CD4 magnetic particles (BD Biosciences). For RNA-seq to analyze the effects of IL-10 on CD4+ T cells (Fig. 5 ), cells were treated by immobilized anti-CD3 antibodies with or without recombinant IL-10 (Peprotech) for 48 h. RNA was extracted with TRIzol reagent (Invitrogen) and purified with the Direct-zol RNA MiniPrep Kit (Zymo Research). Library preparation and high-throughput sequencing were performed at Macrogen using TruSeq RNA Sample Prep Kit v2 and HiSeq 2000 (Illumina) or GeneWiz using the NEBNext Ultra II Directed RNA library kit (New England BioLabs) and HiSeq X (Illumina). RNA-seq data were mapped to the GRCm38/mm10 using HISAT2 (60 (link)). Differently expressed genes were analyzed using HTSEq (61 (link)) and edgeR (62 (link)) or DESeq2 (63 (link)). Volcano plots and heat maps were drawn using ggplot2 (64 ) and gplots (65 ). Expression tiling of RNA-seq was visualized using the Integrative Genomics Viewer (66 (link)). Functional annotation of gene lists with Gene Ontology terms and KEGG pathways was performed with the DAVID tools (29 (link), 30 (link)) and GSEA (44 (link), 45 (link)).
Anti mouse cd4 magnetic particles
Manufactured by BD
Sourced in United States
Anti-mouse CD4 magnetic particles are a laboratory tool used for the isolation and enrichment of CD4-positive cells from mouse samples. These particles are coated with antibodies specific to the CD4 surface marker, allowing for the efficient separation and purification of CD4-expressing cells from complex biological mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant il 10, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hiseq x, by Illumina (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Truseq rna sample prep kit v2, by Illumina (1 mentions)
Propionate, by Merck Group (1 mentions)
Butyrate, by Merck Group (1 mentions)
Anti cd3 mab, by BioXCell (1 mentions)
Anti cd28 mab, by BioXCell (1 mentions)
Dm750, by Leica (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Mouse cd8 t lymphocyte enrichment set, by BD (1 mentions)
Anti cd3 cd28 antibodies, by BD (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Mouse spleen lymphocyte separation medium kit, by TBD Science (1 mentions)
Anti human cd4 magnetic particles, by BD (1 mentions)
5 protocols using anti mouse cd4 magnetic particles
1
RNA-Seq Analysis of IL-10 Effects on CD4+ T Cells
2
Activation of Mouse CD4+ T Cells
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Mouse CD4+ T cells were isolated from spleen or mesenteric lymph node (MLN) using anti-mouse CD4 magnetic particles (Cat#551539, BD Biosciences). CD4+ T cells were seeded in the 24-well plates, and activated with 5 µg/ml anti-CD3 mAb (Clone#145-2C11, Cat#BE0001-1, Bio X Cell) and 2 µg/ml anti-CD28 mAb (Clone#37.51, Cat#BE0015-1, Bio X Cell), or 0.2 million/ml irradiated APCs and CBir1 peptide (ThermoFisher Scientific) in the presence or absence of acetate (10 mM, Sigma Aldrich), propionate (0.5 mM, Sigma Aldrich), or butyrate (0.5 mM, Sigma Aldrich), under neutral (without exogeneous cytokines), Th1 (10 ng/ml IL-12), Th17 (15 ng/ml TGFβ, 30 ng/ml IL-6, 10 µg/ml anti-IFNγ mAb, 5 µg/ml anti-IL-4 mAb), or Treg (5 ng/ml TGFβ and 10 µg/ml anti-IFNγ mAb) polarization conditions. Cells were cultured at 37 °C with 5% CO2. On day 5, the cell yield was about 2 million/ml.
3
CD4+ T Cell Migration Assay
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The method was used as described previously [23 (link)]. Briefly, the A2AR-labeled, empty vector-labeled or blank ZM310 cells were grown on filters as mentioned above for 3 days. The CD4+ T cells were isolated from the spleen with Anti-Mouse CD4 Magnetic Particles (BD, #551539). Then, the medium of the upper chamber was changed to 100 μl 1640 medium containing CD4+ T cells (1 × 106 cells/ml), and the insert was plated into a new well filled with RPMI 1640 (0.5% FBS). Twenty-four hours later, the migrating cells in the lower chamber were subjected to crystal violet staining and counted under a microscope (DM750, Leica).
4
Isolation and Activation of Murine T Cells
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Rab37 knockout (KO) mice were generated as described in Kuo's report 23 (link). CD4+ and CD8+ T cells were isolated from splenocytes derived from wild-type (WT) and Rab37 KO mice using anti-mouse CD4 magnetic particles (BD Bioscience, #551539) and mouse CD8 T lymphocyte enrichment set (BD Bioscience, # 558471) according to the manufacturer's instructions. Cells were cultured in medium containing 10% Fetal Bovine Serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco), and 1% sodium pyruvate (Gibco) and incubated at 37 oC with 5% CO2 in air. Purified naïve CD4+ and CD8+ T cells were stimulated with 2 μg/mL anti-CD3/CD28 antibodies (BD Bioscience, #553057/ #553294) for 24 h or 48 h.
5
Isolation and Purification of CD4+ T Cells
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Fresh human lymphocytes were isolated from peripheral blood, and mouse lymphocytes were obtained from the spleens using the Human Blood Lymphocyte Separation Medium Kit and the Mouse Spleen Lymphocyte Separation Medium Kit, respectively (TBD Science, Tianjin, China). Then, human and mouse CD4+ T cells were sorted by positive selection using Anti-Human CD4 Magnetic Particles and Anti-Mouse CD4 Magnetic Particles (BD Biosciences, San Jose, CA, USA), respectively. The purity of CD4+ cells was generally higher than 95%. Genomic DNA and total RNA of the sorted CD4+ T cells were extracted using DNA/RNA isolation kits (Tiangen Biotech, Beijing, China).
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