Measure signaling cascades in single cells

Phospho-specific antibody flow cytometric analysis of phosphorylated proteins allows researchers a quick and effective way to measure signaling cascades in individual cells. A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular phosphorylated signaling proteins by flow cytometry. Typically, cells are fixed with formaldehyde to stabilize the cell membrane, and then permeabilized with detergent or alcohol to allow antibodies against intracellular antigens access to stain intracellularly.

Validation for phospho-specific antibodies for flow cytometry

Invitrogen eBioscience phospho flow antibodies and related buffer systems are rigorously tested using the methods shown below to help give scientists confidence that they will perform as expected.

Pathway-specific tests

First we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation-specific staining only in the cells in which the specific pathway of interest has been activated.

U937 cells treated as indicated and intracellularly-stained with anti-phospho STAT6 (clone CHI2S4N) APC. Red = untreated, blue = treated.

 

Cell-type specific tests

Second, we verify that phosphorylation-specific staining is observed only in cell types in which the protein is expressed, and not in cell types in which the protein is not expressed. Testing antibodies in pathway- and cell type-specific experiments helps provide end-users with peace of mind regarding the specificity of the antibodies they purchase from us.

Mouse splenocytes were left untreated or were activated with anti-mouse IgM, u chain specific, antibody or with sodium pervanadate. Cells were then surface stained with anti-CD3 (T cells) and with anti-B220 (B cells) and intracellularly stained with anti-phospho-BTK/ITK (M4G3LN) PCP-eFluor 710. Histogram shows levels of phospho-BTK/ITK on CD3+-gated T cells (left) and B220+-gated B cells (right). Orange = unstimulated, purple = +a-IgM, green = +Na3VO4.

 

Daudi B cells (top) or Jurkat T cells (bottom) were left untreated (orange histogram) or were treated with sodium pervanadate (purple histogram). Cells were then intracellularly-stained with anti-phospho-BTK/ITK (M4G3LN) APC (top row) or with competitor’s pBTK/ITK Alexa Fluor 647 (bottom row). Experiment shows that M4G3LN clone sees both BTK (B cells) and ITK (T cells) while competitor’s mAb only sees BTK.

 

Western blotting

Third whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate). Also, whenever possible, we use inhibitors of the pathway to verify that we observe an increase or decrease in signal as appropriate.

Jurkat T cells were left untreated or were treated with P+| +/- MEK1 inhibitor. Western blot shows pERK1/2 signal. Histograms show pERK1/2 under same conditions. Black=untreated, green=PMA+iono, red=PMA+iono+MEK inhibitor. These data show validation of specificity by both western blotting and by flow cytometric analysis.

 

Immunochemistry screening

Immunohistochemistry on FFPE human breast cancer tissue stained with 10 ug/mL MIgG1 Isotype Control Purified (left) and 10 ug/mL Anti-Human NF-kBpS529 (MFCA30) (right). Nuclei are counterstained with hematoxylin. NF-kBpS529 is localized to both the cytoplasm and nucleus of ductal epithelial cells.

 

Buffer compatibility tests

Each phospho-specific antibody has been tested in three different flow cytometry buffer systems: IC Fixation and PermeabilizationFoxp3/Transcription Factor Buffer, and IC Fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for each specific antibody. Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450FITC, and eFluor 660). However, please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. Therefore, we recommend staining after stimulation/treatment, fixation, and methanol treatment. Please refer to our table (Antibody Clone Performance Following Fixation/Permeabilization) to evaluate the performance of numerous clones in the methanol-based buffer system.

Jurkat T cells were untreated (red histogram) or were treated (blue histogram) with H2O2-activated sodium pervanadate for 5 minutes at 37°C.  Cells were then divided and either fixed with IC Fixation Buffer for 10 minutes at RT followed by permeabilization with Permeabilization Buffer (left)fixed and permeabilized using Foxp3/Transcription Factor Buffer for 30 minutes at RT followed by permeabilization with Permeabilization Buffer (middle), or fixed with IC Fixation Buffer for 10 minutes at RT followed by permeabilization with ice-cold 90% methanol for 30 minutes on ice (right).  Cells were then intracellularly stained with eBioscience's pZAP-70/SYK (clone n3kobu5) monoclonal antibody (top row) or with competitor's pZAP-70/SYK monoclonal antibody (bottom row) followed by flow cytometric analysis.

 

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