Protocols
BD™ Phosflow FAQs
What Perm Buffer should I use with my phosflow antibody?
In general, most of the BD Phosflow antibodies will work with Protocol III (Perm
Buffer III) - but it is harshest on cell surface molecules. So if customers
are trying to use Phosflow protocols in combination with cell surface staining,
you should check the link below to see if we have tested the surface marker
of interest, and if it works following the protocol (I, II or III):
http://www.bdbiosciences.com/docs/Validated_Cell_Surface_Markers.xls
Can total and phosphorylated protein be analyzed in parallel?
For Phosflow we recommend to compare 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 customer 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 can not be obtained.
We do have antibodies against total protein (ERK, p38, AKT, STAT, etc) however
we have not validated these with the Phosflow protocols. In general, the epitope
of the antibodies against total protein is not likely to be masked as the
phospho-epitope and therefore, in general, any standard intracellular staining
protocol would work with the anti-total protein. Generally if the antibody
is suitable for Immunofluorescence (IF) it will be likely it will work for
use in flow cytometry. However, please keep in mind we cannot guarantee results
if we have not tested the antibody for use in flow and you will need to test
and optimize the concentration.
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.
Can LWB phosflow samples be stored for a longer time at -20°C OR -80°C?
While fresh cells give the best signal, we have heard from our collaborators
that comparable results have been obtained by fixing the cells, pelleting
the cells, resuspending in PBS (no PFA added) and freezing the cells at -80
for storage. This method has only been tested for storage of several days.
Please note, we cannot guarantee results as we have not extensively tested
this method. We recommend using fresh lysed whole blood with our phosflow
protocols.
What is best protocol to use for phospho-STATs phosflow staining? -I am
getting no staining or high background when staining for any of the phospho-STATS
We can obtain low staining of phospho-stats in cell lines (which have a higher
expression of phospho-Stats) with Perm Buff II (and protocol II), but we obtain
better phospho stat staining with Perm buffer III (558052) (and protocol III).
We have found that staining PBMCs or whole blood for phospho-Stats is more difficult
due to the low expression and donor variability (we have found that phosphorylation
can be donor dependent ). For PBMCs we recommend protocol III and Perm Buffer
III. Often there is a phospho signal present at some basal level even in untreated,
resting or serum starved cells, and this allows for a better indication of the
true difference in expression levels. In the GM-CSF stimulation protocol, we
serum starve the cells overnight.
Can you use the BD Cytofix/Cytoperm buffers in phosflow?
No, our BD Cytofix/Cytoperm buffers were developed for intracellular cytokine
staining. The Perm/Wash Buffer has not been shown to permeabilize the nucleus
and we would recommend our Phosflow Perm Buffers to permeabilize the nucleus.
The Phosflow antibodies have been specificially tested for use with the buffers.
The BD Cytofix Buffer (554655) can be used for protocols II or III, when lysis
of RBC is not required. If you need to lyse RBC, the BD Lyse/Fix Buffer (558049)
should be substituted in.
What protease-free buffer can be used to detach adherent cells?
Protease-free solutions to detach adherent cells (for use in phospho-specific
staining) are an EDTA solution or one of the commercially available products,
such as the enzyme-free Dissociation buffers. Although it is claimed that these
products work better than 'home brew' EDTA solution, none of the trypsin-free
solutions works as well as a trypsin-containing solution. Detaching adherent
cells, especially without using trypsin, can take a long time. In addition,
if cells are activated first, then detached for flow applications, they could
lose phospho-signal 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 completely obliterates surface staining for some antigens (human CD16, 19,
56, 14). In order tot maintain cell surface staining, customers can activate
the cells to induce phosphorylation, fix the cells, stain for surface antigen,
permeabilize the cells with Perm III. Surface staining will decrease in intensity
(separation between negative and positive population will decrease) but it still
will be detectable. This modified protocol has been tested in house with anti-human
CD16, CD14, CD19, CD56
What is the difference in permeabilization between the detergent based Perm
I and the methanol based Perm III?
The detergent based Perm/Wash Buffer I leaves the secondary/tertiary structure
of proteins intact, while methanol based Perm Buffer III disrupts them. This
disruption helps for Stats that are dimerized, but might not be necessary for
a cytosolic protein that isn't bound to anything else via its phosphoepitope.
Can I costain with CD127 and phosflow antibodies?
No, CD127 is not compatible with the Perm Buffers I, II, and III.
Can I stain for FoxP3 and phosflow antibodies?
Yes, with Perm Buffer I, you will need to gate on the CD4+CD25+bright population,
to see FoxP3 minimally if separated out from CD4 or CD25. Please note, you may
not be able to visually separate out FoxP3 marker from CD4 brightCD25bright
using BD Phosflow Perm Buffer (I, II,III) without this gating strategy. In addition,
if you were to look at FoxP3 versus CD4 or CD25, you will not be able to separate
out FoxP3 from these populations. Generally the detergent based, Perm Buffer
I will work better for foxp3 staining than the methanol based Perm III.
What is the usual cell density/volume we are working with for the mouse
splenocyte BD Phosflow protocols?
Depending upon the experiment, around 4 million cells per ml, in a volume
of 2 ml. Customers need to be aware that the density may vary, sometimes more
or less is better.
What mouse model was used with pStat5 (pY694), clone 47, for BD Phosflow
staining?
We have tested on mouse spleen cells. For Stat 5 activation, we used GM-CSF.
The activation is observed mainly on CD11b+ cells.
Why do you suggest a blocking step for a phosflow protocol that requires
activation by anti-human CD3 antibody (UCHT1, cat# 555329) and anti-human
CD28 antibody (CD28.2, cat# 555725)?
Phosflow protocol: Cells that were stimulated by cross-linking of CD3 and
CD28 with NA/LE Mouse anti-Human CD3 mAb UCHT1 (Cat. No. 555329) and NA/LE
Mouse anti-Human CD28 mAb CD28.2 (Cat. No. 555725) on ice for 15 minutes followed
by Purified Goat anti-Mouse Ig(Cat. No. 553998) on ice for 15 minutes (for
cross linking), and then allowed to undergo phosphorylation at 37°C for 3
minutes. The cells were fixed (BD Cytofix™ Buffer, Cat. No. 554655) for 10
minutes at 37 °C, permeabilized (BD™ Phosflow Perm Buffer III, Cat. No. 558050)
on ice for at least 30 minutes, blocked with normal mouse immunoglobulin,
and then stained with phosflow antibodies. We suggest doing blocking with
normal mouse immuniglobulin to block Goat anti-Mouse Ig that was left unbound.
R&D routinely uses CALTAG Cat#10400 at 10ug/test/100ul and incubate at RT
for 10min.
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