In vitro assay: For intracellular staining, Brefeldin A (Golgi Plug) was added for the last 6 hours of 72-hour co-culture. After centrifugation, the supernatant was collected for ELISA assays. Cells were collected for flow cytometry. In vivo assay: tissue was homogenized in 2% FBS RPMI and filtered by a 40 μm strainer. The cells were stained following the manufacturer’s protocol. For phospho-signal flow cytometry, we use BD Phosflow Fix Buffer and BD Phosflow Perm/Wash Buffer III. All the procedures followed by BD™ Phosflow Protocols for Mouse Splenocytes or Thymocytes. All samples were acquired with Beckman CytoFLEX LX Flow Cytometer and analyzed with FlowJo software.
Phosflow protocols for mouse splenocytes
Manufactured by BD
The BD™ Phosflow Protocols for Mouse Splenocytes is a lab equipment product designed for the analysis of phosphorylated proteins in mouse splenocytes using flow cytometry. It provides a standardized protocol and reagents for the preparation, fixation, and permeabilization of mouse splenocyte samples prior to intracellular staining and analysis.
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2 protocols using phosflow protocols for mouse splenocytes
1
Intracellular and Phospho-Signal Cytometry
2
Phosphoflow Protocol for Lung Cells
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Lung tissue was processed as described above, except that the digestion with 2 mg mL -1 of collagenase IV was reduced to 30 min at 37 °C in a shaker. Phosphoflow staining was performed according to Protocol I described in BD Phosflow Protocols for Mouse Splenocytes. Briefly, following viability dye staining, samples were fixed with BD Phosflow Lyse/Fix Buffer for 11 min at 37 °C, permeabilized with BD Phosflow Perm/Wash buffer I for 30 min at RT, then stained with antibody mixture for 1 h at RT.
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