B cells stained with flow cytometry mAbs to CD20, CD27, CD21, IgG3, and IgD (identified above) were stimulated with 10 μg/ml goat F(ab’)2 anti-IgM (Jackson ImmunoResearch Laboratories; Cat# 109–006-129) at 37 °C for 2 min. For the detection of phosphorylated signaling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and were stained separately with PE-conjugated mAbs to Syk phosphorylated at Tyr348 (clone I120–722), Btk phosphorylated at Tyr223 (clone N35–86) and PLC-γ2 phosphorylated at Tyr759 (clone K86–689.37) (all from BD Biosciences). Flow cytometry was performed on an LSRFortessa.
Cytofix and phosflow perm wash buffers
Manufactured by BD
BD Cytofix and Phosflow Perm/Wash buffers are reagents designed for use in flow cytometry applications. They are used to fix and permeabilize cells to enable intracellular staining and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Goat f ab 2 anti human iga g m, by Jackson ImmunoResearch (3 mentions)
Aurora cytometer, by FlowJo (3 mentions)
Alexa fluor 488 conjugated mab against phosphorylated, by BD (2 mentions)
Alexa fluor 647 conjugated mab against phosphorylated, by BD (2 mentions)
Goat f ab 2 anti igm, by Jackson ImmunoResearch (1 mentions)
Pe conjugated mabs, by BD (1 mentions)
Goat anti rabbit igg pe, by Thermo Fisher Scientific (1 mentions)
F ab 2 goat anti human igm, by Jackson ImmunoResearch (1 mentions)
5 protocols using cytofix and phosflow perm wash buffers
1
Flow Cytometry Analysis of BCR Signaling
2
Phosphorylation of Syk and PLC-γ2 in BCR-stimulated PBMCs
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PBMCs stained with mAbs against CD19, CD20, CD3, CD27, CD21, IgD, and BV421-conjugated RBD-B.1 were resuspended in RPMI 1640–10% FBS and stimulated with anti-BCR as described previously (Kardava et al., 2018 (link)), with the following modifications. The cells were stimulated at 37°C for 2 min with 10 μg/ml goat F(ab’)2 anti-human IgA/G/M (# 109-006-064 from Jackson ImmunoResearch Laboratories). For the detection of phosphorylated signalling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and stained with Alexa Fluor 488-conjugated mAb against phosphorylated Syk (p-Y348) and Alexa Fluor 647-conjugated mAb against phosphorylated PLC-2 (p-Y759) (BD Biosciences). The samples were acquired on an Aurora cytometer and analysis was performed by FlowJo V10.
3
Phosphorylation of Syk and PLCγ2 in BCR-Stimulated PBMCs
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PBMCs stained with mAbs against CD19, CD20, CD3, CD27, CD21, IgD, and BV421-conjugated RBD-B.1 were resuspended in RPMI 1640-10% FBS and stimulated with anti-BCR as described previously (Kardava et al., 2018 (link)), with the following modifications. The cells were stimulated at 37°C for 2 min with 10 μg/ml goat F(ab’)2 anti-human IgA/G/M (# 109-006-064 from Jackson ImmunoResearch Laboratories). For the detection of phosphorylated signaling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and stained with Alexa Fluor 488-conjugated mAb against phosphorylated Syk (p-Y348) and Alexa Fluor 647-conjugated mAb against phosphorylated PLCγ2 (p-Y759) (BD Biosciences). The samples were acquired on an Aurora cytometer and analysis was performed by FlowJo V10.
4
Phosphorylation of B Cell Signaling Intermediates
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Five unrelated healthy adult volunteers were studied, each on a different day. Peripheral blood B cells were isolated as described in the Cell purification and cell culture conditions for in vitro treatment section. For phosphorylation assays, glucocorticoid- or vehicle-treated B cells were stained with mAbs against CD19, CD27, CD10, and IgD, as detailed in the Assessment of B cell surface markers section, then stimulated with 10 µg/ml goat F(ab′)2 anti-human IgM (Jackson ImmunoResearch Laboratories) at 37°C for 2 min. For the detection of phosphorylated signaling intermediates, cells were fixed and permeabilized using BD Cytofix and Phosflow Perm/Wash buffers (BD Biosciences) and stained separately with a PE-conjugated mAb against phosphorylated PLC-γ2 (pY759) clone K86-689.37 (BD Biosciences), or an unconjugated polyclonal antibody against phosphorylated CD79A(Tyr182) (Cell Signaling Technology; cat. no. 5173) followed by staining with secondary goat anti-rabbit IgG-PE (Thermo Fisher; cat. no. P-2771MP). Flow cytometric analyses were performed as described above.
5
BCR Stimulation and Signaling Analysis
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