Summary Cyclin B1 help
astbr at helix.nih.gov
Fri Feb 20 10:42:30 EST 1998
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:
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
I would greatly appreciate any help and post 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.
From: "Telford, William Ph.D." <TelfordW at hss.edu>
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
Hope this helps.
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
From: "Michael G. Ormerod" <Michael_Ormerod at compuserve.com>
Subject: cyclin B1 Help!!
Sender: "Michael G. Ormerod" <Michael_Ormerod at compuserve.com>
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.
34 Wray Park Road
Reigate RH2 ODE
Telephone: voice & FAX: (0)1737 241726
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
From: "Steffen Roth" <steffen.roth at rhein-neckar.de>
NeoMarkers sells the Cyclin B1 reactive clone V152 and a polyclonal
antibody. The datasheets (WORD-6 format) are available at:
V152's datasheet: 338.zip
polyclonal's datasheet: 008.zip
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