SIMULTANEOUS SURFACE AND PI STAINING QUESTION

Dear flowers,

 

Thank you very much for your replies. Further below is a compilation of
them.

 

Merry Christmas to everybody.

 

 

Dear flowers,

 

 I have a couple of questions regarding simultaneous surface  and cell
cycle analysis with PI.

 I stained a lymphoblastoid cell line with FITC and APC conjugates,
subsequently

fixed it with 70% ethanol and then incubated it with RNAse and PI.

I acquired the cells in a FacsCalibur (settings set for cell cycle
analysis, PI is read on FL2-A) and I saw

a strange picture of double positive events at the FL1 vs FL4 plot.

I repeated the acquisition with my usual four color settings

and I've noticed that there was no positivity for APC, while my unfixed
sample was

all double positive for both FITC and APC.

 

1) Does the fixation destroyed the APC epitope (if yes, then why not
also the FITC)?

 

2) Can I use FL4 and FL1 in Log scale for acquisition and analysis,
while I perform cell cycle analysis with PI?

 

Thanks in advance,

 

 

 

 

Dear Ioannis,

 

Concerning your questions:

1.I never performed ethanol fixation after APC staining, but ethanol
might affect APC, just like it affects R-PE, published 12 years ago by
Ken Toba in Cytometry 1992;13(1):60-7. "Improved staining method for the
simultaneous flow cytofluorometric analysis of DNA content, S-phase
fraction, and surface phenotype using single laser instrumentation." We
observed exactly the same.

2. It is no problem using log amps for FITC and APC.

3. It might be possible that you are dealing with doublets. For a more
optimal discrimination between singlets and doublets using PI and APC as
fluorescent dyes for labelling DNA and a protein, respectively, you need
to change the hardware of the Calibur, since APC is measured on the FL4
at the costs of the pulse-width (FL1, FL2 or FL3). The solution is quite
simple. You only need a new connecting wire (can be obtained from BD)
and make some disconnections and new connections in the Calibur. The
protocol was published in the following paper: "Four-color
multiparameter DNA flow cytometric method to study phenotypic intratumor
heterogeneity in cervical cancer.
Cytometry. 2000 Feb 1;39(2):96-107."

 

Good luck,

 

Willem Corver

 

Yes, APC is sensitive to EtOH fixation.  Try FITC, PE conjugates (PE,
PECy5, PECy7) or Pacific Blue, if you can.

 

  APC, PerCP and their conjugates are all sensitive to EtOH fixation.

 

R. Friedline
UMass Medical School

 

would suggest de convoluting and finding out if your Mabs and antigens
do make it through the fixation steps.	i.e. stain things independently
and in combinations and then run them with and with out fixation and
with and with out PI post fixation.

 

then you will know for sure

 

Pb

 

Which epitopes were you trying to detect with the mAbs? Some antigens
are less resistant to ETOH fixation than others.  The best approach is
to prepare the samples again without fixation and analyse the
fluorescence then fix as you did before but don't add PI and run them to
check that the two epitopes are detectable. If this works then take some
non-labelled cells, fix and add PI and then analyse with the same
instrument settings and check for signals in FL1 and FL4 (make certain
that the time delay calibration for the second laser is correctly set up
- see the manual for instructions).

Call me on the number below if I can be of any more help.

Regards

Mark

 

 

 

Dr Mark W Lowdell PhD MRCPath

Senior Lecturer in Haematology / 

Director of Laboratory of  Cellular Therapeutics

Tel: 020 7830 2183

Fax: 020 7830 2092

 

Dear Loannis,

 

I hae been doing some experiments with 4 color staining of mouse bone-
marrow cells and have experienced the same troubles you did but I use 7-
AAD instead of PI. 7-AAD has the advantage that you don't need RNA-se 

and that it can be used beside FITC, PE and APC. I have made a four
color 

staining protocol with Alexa 488, PE, 7-AAD and APC.

I also experienced the removal of APC epitope but when I started to
fixate 

my cells with 0,1% paraformaldehyde for 5-10 min and the with 0,01% 

saponine for 15 min whereafter I stained with 7-AAD i got good picturs.
The 

dissadvantage of my protocol is the fact that it's stained with a 

phosphatecitrate buffer which has a pH of 4,7 where the fitc signal is
very 

bad. 

response to question 1: yes I believe that fixation can destroy APC
epitope. 

Why not fitc, not sure maybe it's less sensitive. I think that every
kind of 

cell/tissue has an optimal fixation methode. 

2) sure that FL4 and FL1 can be used with analysis fo PI but fixation 

protocol is very important.

 

If it's possible to replace the FITC for maybe Alexa488 or PE you could
try 

my protocol for you cells.  If so, just let me know and I will mail it 2
you.

 

Hope to hear from you and good luck!!

 

Kyrjon van Pelt

 

Drs. A.P. van Pelt

 

Department of Hematology, 

Internal Medicine, Academic Hospital Groningen

Tel: +31 50-3632743/050-3611437

 

Hi Ioannis,

 

FITC survives ethanol fixation because it is a small molecule and
fluorescence is maintained after the dehydrating effect of the alcohol.
APC, despite being a much bigger molecule, should also survive ethanol
fixation and we have used this successfully here. PI will fluoresce
equally as brightly in FL3 as FL2 on a Calibur and that signal may also
spill into FL4 and this may be what you are seeing - I would need to see
a plot (or better a data file) to assess that.

 

You can certainly also use log scaling for APC and FITC (this is what we
do during the BrdU technique) - because of the disparity between the
relative strengths of the signals from the DNA dye and the other
fluorochromes there are few compensation problems other than the PI
fluorescence emission.

 

The proof of the survival of fluorescence is running the samples pre-
and post-fixation and you should see the same percentages positive
(autofluorescence levels may increase). If the levels drop you may wish
to consider alternative methods - we have had success with a brief PFA
fix and then use of saponin to permeabilise the cells to get the DNA dye
in - in this way we have used FITC, PE and APC alongside 7AAD as our DNA
dye.

 

Hope that helps - good luck!
Derek

 

 

-- 

FITC conjugated Abs tolerate EtOH fixation very well but PE fluorescence
is completely destroyed - presumably due to denaturing.  I would assume
APC responds in a similar manner to PE.

good luck, TomD

 



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