Summary Cyclin B1 help

Andreas Steinbrecher astbr at helix.nih.gov
Fri Feb 20 10:42:30 EST 1998


Hi all,
I was very happy to get all  the responses and suggestions posted below. This
was the first time I used this website and it was great!
Thank you very much.

First the problem:
Hi all,
we are trying to double-stain ConA stimulated murine T cells with PI and
Pharmingens monoclonal ab against cyclin B1 using Pharmingens protocol for
cyclin staining (fixation in 75% ETOH, -20°C for at least several days, 0.25%
triton x-100 for 5 min, incubation with mab for 30 min at RT, incubation with
PI/RNAseA for 30-45min before analysis). As this anti-human ab is supposed to
be crossreactive for mouse cyclin B1 but has not been tested with murine cells
for flow cytometry by Pharmingen  I used human PHA stimulated PBMC as a
control. However in neither cell type do I see consistent cyclin B1 staining.
Sometimes in the human cells there is very little that is the present during
the whole cycle and probably non-specific.
Does anybody have experience with this antibody and this protocol? With murine
cells and cyclin staining in general? Does anybody know a better antibody for
this purpose? 
I would greatly appreciate any help and post the answers.
Andreas

The answers:

From: Padma K Narayanan @ SB_PHARM_RD 
Date: 18-Feb-98 02:10:45 PM 
The Santa Cruz antibody worked pretty well for me. I would also adopt a 
different fixation procedure. The details are in the paper published in 
Experimental Cell Research. The details followNarayanan, P.K., Rudnick, 
J.M., Walthers, E.A., and Crissman, H.A. (1997) Modulation of cell cycle 
and cyclin B1 expression in irradiated HeLa cells and normal human skin 
fibroblasts treated with staurosporine and caffeine. Experimental Cell 
Research. 233, 118-127. 
Good luck 
Padma

From: "Telford, William Ph.D." <TelfordW at hss.edu> 
To: cyto-inbox
Subject: cyclin B1 antibodies 
Date: Wed, 18 Feb 98 12:05:00 PST 
Which cyclin B1 antibody from Pharmigen are you using, clone GNS-1 (cat. no. 
14541A) or clone GNS-11 (14551A)? We have tried both for flow and only 
GNS-1 works in our hands for human cells. I don't know if it crossreacts 
with mouse.
Hope this helps.
Bill
No, we've tried the Pharmingen protocol (as described by Dr. Darzynkiewicz) 
and it works. In fact, we have found B1 labeling to be much less 
technique-sensitive than the E and D cyclins - straight ethanol fixation at 
70% has worked fine. We have used HL-60 cells as our control line. The 
only difference I can think of is the choice of fluorochrome - for some cell 
types, FITC - cyclin B1 labeling has been quite dim in our hands. For these 
cases, we use anti-cyclin B1 and a Cy5-anti-mouse IgG (from Caltag or 
Jackson ImmunoResearch) using the diode laser on our FACSCalibur or the HeNe 
laser on our Vantage. It is a lot brighter than FITC. An example of FITC 
and Cy5 comparison for cyclin detection is on our website 
(<http://www.hss.edu/research/flow/4color.htm>http://www.hss.edu/research/f
low/4color.htm).
Bill

From: "Michael G. Ormerod" <Michael_Ormerod at compuserve.com> 
Subject: cyclin B1 Help!! 
Sender: "Michael G. Ormerod" <Michael_Ormerod at compuserve.com> 
To: cyto-inbox
Cc: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu> 
When I used the Pharmingeb ab to cyclin B1, I fixed in 70% ethanol at -20C, 
stored at -20C for 1 h to 2 days, brought to PBS, 5% BSA, 0.1% Triton X, 
incubated with monoclonal ab overnight on ice, washed in the same buffer, 
finally resuspended in PBS, PI, RNase, incubated 30 min at 37C and ran. 
Using a human lympoblastoid cell line, I obtained excellent results. Anyone 
who has a copy of my CD-ROM will find the listed data files thereon.
Michael Ormerod 
34 Wray Park Road 
Reigate RH2 ODE 
Telephone: voice & FAX: (0)1737 241726 
Web site:
<http://ourworld.compuserve.com/homepages/Michael_Ormerod>http://ourworld.co
mpuserve.com/homepages/Michael_Ormerod

From: "James W. Jacobberger" <jwj at po.cwru.edu> 
Organization: Case Western Reserve University 
I don't know whether this reacts with mouse or not, 
but we obatined very good results with human cell lines. 
However, we used fixation with 90% MeOH at -20degC. 
When staining and washing. be sure to use a blocking reagent like 
BSA or serum. I don't know much about PBMC but you should pick 
a control with a significant number of G1+M cells since 
you won't see much staining until late S and G2+M.
hope this helps 
jwj

From: "Steffen Roth" <steffen.roth at rhein-neckar.de> 
To: cyto-inbox
NeoMarkers sells the Cyclin B1 reactive clone V152 and a polyclonal 
antibody. The datasheets (WORD-6 format) are available at: 
http://www.labvision.com/datasheet/
V152's datasheet: 338.zip 
polyclonal's datasheet: 008.zip
Best regards 
Steffen






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