The spleen was removed from the infected mice, and the spleen lymphocytes were separated using the Mouse Spleen Lymphocyte Separation Medium Kit (TBD Science, Tianjin, China) according to the manufacturer’s instructions. Separated lymphocytes and recombinant pMV262/MS, Rv0888NS/MS, D438A/MS, and H481N/MS were co-incubated overnight. IL-6, TNF-α, IL-1β, and IFN-γ levels in the supernatant were detected with a commercially available mouse IL-6, TNF-α, IL-1β, and IFN-γ ELISA Kit (eBioscience, Waltham, MA, USA) according to the manufacturer’s instructions.
Mouse spleen lymphocyte separation medium kit
Manufactured by TBD Science
Sourced in China
The Mouse Spleen Lymphocyte Separation Medium Kit is a laboratory product designed to isolate mouse spleen lymphocytes from whole spleen tissue. The kit contains a density gradient medium that allows for the separation of lymphocytes from other cell types during centrifugation.
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4 protocols using mouse spleen lymphocyte separation medium kit
1
Immune Response Assay in Spleen Lymphocytes
2
Phenotypic Analysis of Mouse Lymphocytes
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Mouse lymphocytes were extracted from the fresh mouse spleens using the Mouse Spleen Lymphocyte Separation Medium Kit (TBD Science) and were stained with anti-CD4-FITC (BD Biosciences). To induce secretion, the lymphocytes were stimulated with Leukocyte Activation Cocktail (BD Biosciences) for 6 hours. After surface marker staining with anti-CD4-FITC (BD Biosciences), lymphocytes were permeabilized and incubated with anti-IFN-γ-APC, anti-IL-4-PerCP-Cy5.5, anti-FOXP3-APC, and anti-IL-17A-PerCP-Cy5.5 (BD Biosciences). Flow cytometry was performed on a BD FACSCalibur flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (version 10.0.7; Treestar, Ashland, KY, USA).
3
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).
4
Isolation and Identification of Murine Tregs
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Single-cell suspensions of lymphocytes were extracted from fresh spleens tissue using Mouse Spleen Lymphocyte Separation Medium Kit (TBD Science, Tianjin, China). Single-cell suspensions of lymphocytes were stained for Treg cells using Mouse Regulatory T Cell Staining Kit (eBioscience, San Diego, CA, USA) with anti-CD4-FITC, anti-CD25-APC, and anti-FOXP3-PE according to the manufacturer's instructions. Flow cytometry was performed and analyzed on BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).
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