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Intracellular Flow and Phosflow FAQ

Which perm buffer should I use with my BD Phosflow antibodies?

In general, most BD™ Phosflow antibodies are compatible with Protocol III (BD Phosflow Perm Buffer III). This buffer has the harshest effect on cell-surface molecules, so investigators using BD Phosflow protocols in combination with cell-surface staining should check the link below for antibodies that have been validated with BD Phosflow Protocols-IV.

Can total and phosphorylated protein be analyzed in parallel?

For BD Phosflow applications, we recommend comparing the basal phospho-protein level from an unstimulated sample (the negative control) against the phospho-protein content of the stimulated sample, rather than comparing the content of total protein and phospho-protein together in the same sample. In some cases, however, investigators may be interested in analyzing the total content of a specific protein (for example, when comparing different human tumor samples for which a negative equivalent cannot be obtained). We have antibodies against total proteins (ERK, p38, AKT, Stat, etc) but have not validated these for BD Phosflow protocols. In general, antibodies recognizing epitopes against a total protein are unlikely to recognize a specific phospho-epitope. Therefore, any standard intracellular staining protocol should be suitable with the total protein antibody. If the antibody is suitable for immunofluorescence (IF), it will likely be compatible for flow cytometry. However, please keep in mind we cannot guarantee results if we have not tested the antibody for use in flow cytometry and we recommend that investigators validate the reagent under their experimental conditions.

Is there a preference for using EDTA or heparin in Phosflow protocols?

Generally, there is no preference between EDTA and heparin, and we do not have a specific list of which protocols are affected by the type of anti-coagulant. However, heparin would be optimal in activation conditions requiring Ca++, as EDTA is a chelator and will deplete the Ca++. However, in cases where PMA/ionomycin is used for stimulation of whole blood, EDTA gives better cell separation between monocytes and granulocytes.

Can lysed whole blood BD Phosflow samples be stored for a longer time at -20°C - -80°C?

While fresh cells provide the greatest signal-to-noise ratio with BD Phosflow antibodies, we have heard from our collaborators that comparable results have been obtained by fixing cells, pelleting cells (per the recommended BD Phosflow protocols), then resuspending cells in PBS (without paraformaldehyde) and freezing the cell/PBS mixture at -80°C for storage. This method has only been tested for storage of several days. Please note, we have not extensively tested this method within BD Biosciences and the use of fresh lysed whole blood is recommended with BD™ Phosflow protocols.

Which BD Phosflow protocol is recommended for phospho-(p)Stat antibodies? I am getting no staining or high background when staining for any of the pStat proteins.

We obtain brighter pStat staining with BD Phosflow Perm Buffer III (Cat. No. 558050, and Protocol III) and observe lower staining of pStats in cell lines (which have higher expression of pStats) with BD Phosflow Perm Buffer II (Cat. No. 558052, and Protocol II). In human cells, we have found that staining PBMCs or whole blood for pStats is more difficult due to low expression and donor variability (we have found that phosphorylation levels can be donor-dependent). For PBMCs, we recommend Protocol III and Perm Buffer III.  Often, pStat signals are present at basal levels in untreated, resting, or serum-starved cells, and this can provide an indication of the true difference in expression levels compared to a treated sample. For example, when stimulating with GM-CSF for phospho-Stat5 expression, we serum-starve the cells overnight.

Can BD Cytofix/Cytoperm buffers be used in BD Phosflow applications?

No, BD Cytofix/Cytoperm™ buffers were developed for intracellular cytokine staining and BD Phosflow antibodies have been specifically tested for use with the BD Phosflow buffers. BD Perm/Wash™ Buffer (Cat. No. 554723) included in BD Cytofix/Cytoperm™ kit (554714) has not been shown to permeabilize the nucleus, so we recommend using BD Phosflow perm buffers. BD Cytofix™ fixation buffer (Cat. No. 554655) can be used for fixation in Protocols II or III when lysis of RBCs is not required. If RBC lysis is required, the use of BD Phosflow Lyse/Fix Buffer (Cat. No. 558049) is recommended.

Which protease-free buffers can be used to detach adherent cells?

Protease-free solutions to detach adherent cells (for use in BD Phosflow applications) are EDTA solutions or commercially-available products such as enzyme-free dissociation buffers. Although it is claimed that these products are more effective than 'home brew' EDTA solutions, none of the trypsin-free solutions works as well as a solution containing trypsin. Detaching adherent cells, especially in the absence of trypsin, requires longer incubation times. In addition, if cells are activated first, then detached for flow cytometry applications, they could lose phospho-signals very quickly.

What is the best way to maintain cell-surface staining with Perm Buffer III?

Many times the regular fixation/permeabilization protocol with Perm Buffer III (Cat. No. 558050) completely obliterates surface staining for some antigens such as human CD16, CD19, CD56, and CD14.

What is the difference in permeabilization between the detergent-based and the methanol-based Perm buffers?

The detergent-based BD Perm/Wash buffers generally leave the secondary/tertiary structure of proteins intact, while methanol-based Perm buffers disrupt structural integrity. This disruption helps for Stat proteins that are dimerized, but may not be necessary for a protein that is not bound to anything else via its phospho-epitope.

Can I co-stain with CD127 and BD Phosflow antibodies?

No, CD127 is not compatible with Perm Buffers I, II, or III.

Can I stain for FoxP3 and BD Phosflow antibodies?

Yes, using Perm/Wash Buffer I and strategic gating of the CD4+CD25+bright population, we can see the FoxP3-positive population separated from CD4 or CD25. Generally, the detergent-based, Perm/Wash Buffer I will work better (in terms of signal to noise) for FoxP3 staining than the methanol-based Perm Buffer III.

What is the usual cell density/volume we are working with for the mouse splenocyte BD Phosflow protocols?

Depending upon the experiment, we use around 4 million cells per mL, in a volume of 2 mL for stimulation. We recommend performing timepoints (for example, 5, 10, 15 minutes) to find the optimal stimulation conditions. We recommend optimizing activation conditions for your application.

Why do you suggest a blocking step for a Phosflow protocol that requires activation by anti-human CD3 antibody (UCHT1, Cat. No. 555329) and anti-human CD28 antibody (CD28.2, Cat. No. 555725)?

We suggest blocking with normal mouse immunoglobulin (Ig) to block remaining unbound goat anti-mouse Ig from the activation step. We routinely use normal mouse Ig (Invitrogen, Cat. No. 10400C) at 10µg/test/100µl and incubate at RT for 10 minutes. (For protocol, see BD Phosflow™ Protocols for TCR Stimulation)

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