Thymi, spleen and Peyer’s patches (PPs) were isolated from mice. Spleen and thymi single-cell suspensions were generated as described above. PPs were pressed through a 70-μm nylon screen to generate a single-cell suspension. Single-cell suspensions were stained for 30 min at 4 °C with the indicated antibodies. Staining for CD40L was performed as previously described (58 (link)). For intracellular Ig H + L staining, the BD Cytofix/Cytoperm kit was used as per the manufacturer’s instructions. For intranuclear transcription factor staining, the Tonbo Foxp3 / Transcription Factor Staining Buffer Kit was used as per the manufacturer’s instructions. Samples were acquired with a BD LSRII or Fortessa X-20 (BD Biosciences) and analyzed with FlowJo version 10.8.1 (FlowJo LLC).
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Multi-tissue Immune Cell Isolation
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Corresponding Organization :
Other organizations : University of Minnesota Medical Center, Rutgers, The State University of New Jersey
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Variable analysis
independent variables
- Isolation of Thymi, spleen and Peyer's patches (PPs) from mice
- Single-cell suspensions of spleen, thymi, and PPs
- Staining of single-cell suspensions with indicated antibodies
- Staining for CD40L
- Intracellular Ig H + L staining
- Intranuclear transcription factor staining
- Staining duration of 30 min at 4 °C
- Use of BD Cytofix/Cytoperm kit for intracellular Ig H + L staining
- Use of Tonbo Foxp3 / Transcription Factor Staining Buffer Kit for intranuclear transcription factor staining
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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