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Re: Summary of GFP/YFP Responses (13)
Posted by: ckuszyns@UNMC.EDU
Charles A. Kuszynski,
Ph.D.
Assistant Professor/Director
Cell Analysis Facility
University of
986495
402 559-6267 voice
402 559-4077 fax
ckuszyns@unmc.edu
First I would like to thank all of thr respondents for their input. We are able to get separation of these two
with the suggested filter set. We do however
see a lot of noise with the 510 filter.
We are ordering a new one.
The following are the responses I
received.
We sort GFP/YFP transfected
cells on our Vantage SE using 488 nm excitation (200 mW) and measured in the FITC & PE channels. The filters
and beamsplitter are a little different and we
compensate between the two, but it is generally straight-forward. I could see this
having problems if there was very low levels of expression.
Marty
Hi Charles. We routinely run GFP/YFP double
detection on a 2 laser MoFlo here in London. I've attached
a ppt showing the various optical setups you need to
consider. I usually run with option 1, but I have tried all three, with very
little difference between them. The most critical things to have are GFP and
YFP single controls, otherwise compensation can be very very
tricky. The last slide shows what you're aiming for, compensatable
dual expression.
Hope it helps
Wayne Turnbull
Flow Cytometry Services
Peter Gorer
Department of Immunobiology
3rd Floor New Guy's House
GKT Guy's Hospital
I (and I'm sure many others also) have
successfully sorted GFP / YFP samples on a stream in air sorter. Specifically, on a MoFlo
with an Enterprise II laser emitting 150 mW @488
nm. The optical set up was a 510 / 21
for the GFP channel, a 550 / 30 for the YFP channel, and a 530 DMSP splitting
the two. PMT voltages were relatively
low for both channels ~420
V), and the compensation pretty high:
36% to subtract GFP from the yellow channels, 85% to subtract YFP from
the green channels. GFP separation was a
full 3 logs, YFP separation was 2.5 logs.
NB: just a detail, but because I
used a DMSP, FL1 was yellow and FL2 was green.
That would be the same set up on the FSP if you used this filter
combination.)
Briefly:
it can be done on a jet-in-air; I found the separation at 488 nm to be
quite satisfactory, but 457 nm should be even better if you can get enough
power. Compensation with the 510 and 550
filters is a bit tricky to set because they are relatively high, but this is
dependent on relative PMT voltage. (A
the risk of stating the obvious: set the
comps in the range I mentioned, then adjust PMT voltages until the negatives
and single positives fall where they should.
I suggest this method because if the PMT voltages are too far off, no amount
of compensation will suffice.)
Hope this helps,
Claude
I assume that you have the correct filter
set. For FL1, I put in a 510/21 band pass filter for GFP measurements. I then
insert a 550/30 band pass in FL2 for YFP measurements. I place a 525 dichroic
filter in the FL1/FL2 dichroic spot. (Omega sells these filters in a set if you
don't have them all). I can't imagine why the manufacturer would recommend for
you to use 457 nm. That wavelength will not excite YFP (it's hitting only ~7%
of its excitation spectrum, whereas the 488 line hits it at ~35%).
Hope this helps.
C. William King, Jr.
"Technical Coordinator Flow
Cytometry" - whatever that means
FACS Core Research Facility
Children's Research Institute
Children's
202.884.2308 tel
202.884.5808 fax
wking@cnmc.org
We have done this on a FACStar Plus stream-in-air instrument using the filter set
recommended by Omega (I think, but it could have been Chroma). Works fine, with 488nm excitation
and not too much compensation required.
Alice L. Givan
Englert Cell Analysis Laboratory
tel 603-650-7661
fax 603-650-6130
givan@dartmouth.edu
Charles
I've only done CFP/YFP. I guess that your
FACSstar doesn't have dual lasers, since it's not a "PLUS"?
457 is great for the CFP (it's what I use), but it's just so weak for YFP. YFP
is best excited by the 514 nm line.
<<http://www.bdbiosciences.com/spectra>>
What about changing the filters to 510/20
and 550/30:
Stull RA, Hyun WC, Pallavicini
MG. Simultaneous flow cytometric analyses of enhanced green and yellow fluorescent
proteins and cell surface antigens in doubly transduced
immature hematopoietic cell populations. Cytometry. 2000 Jun 1;40(2):126-34.
PMID: 10805932
Dennis J. Young
Flow Cytometry Core Facility
Bldg #4, Room 126
Mail:<<mailto:djyoung@ucsd.edu>>
WWW:<<http://cancer.ucsd.edu/flow/>>
Telephone:(858) 822-0407 FAX: (858) 822-0403
Bill Telford(NCI)
and Theresa Hawley (ARC) have successfully done this with a FACSVantage
SE (see http://home.ncifcrf.gov/ccr/flowcore/index.htm under projects). Also see Beavis and Kalejta (Cytometry 37:68-73, 1999) for a full description.
Joanne Lannigan,
MS
Director, Flow Cytometry Core Facility
Jordan Hall, Room 7067
Office: 434-924-0274
Lab: 434-243-2695
Fax: 434-982-1071
email: joannelannigan@virginia.edu
We do this routinely on our Vantage SE
and DiVa. You'll need some different filters. Replace
the normal FL1/FL2 splitter (probably a 560 sp) with a 530 sp. Use a 510/10 filter
on FL1 to detect GFP, and a 550/30 filter on FL2 to detect YFP.
We run GFP/YFP samples on an FL1 vs. FL2
dot plot without compensation. The populations usually neatly separate
themselves from the background.
Dax Arguello
The J. David Gladstone Institutes
Flow Cytometry Core Facility
Building 3, Room 514
Hi Charles -
Did this on a MoFlo -- HEK293 transients of GFP and YFP (separately) for the
first one, then GFP stables and YFP transients on the second.
500-510 3rd millenium bandpass filter (omega) gives poor CV's, but allows for
plenty of signal.
These were both generated off of the 488
line.
Let me know if you have any questions.
Andrew BEERNINK
(See attached file: GFP-YFP overlay.bmp)(See attached file: 505-540 overlay.bmp)
Hi, Dr. Kuszynski,
With a optical configuration shown in the
Fig.1 of our Cytometry paper (Part A 53A: 39-54, 2003), the CFP, GFP,YFP and dsRFP can be successfully
detected simultaneously on FACSDiVa using 458 nm
excitation. GFP signal collected in FL1 using 510/10 nm filter was separated
from YFP, which was measured in FL3 with 530/30 filter, by two sequential
dichroic filters 530 SP and 500 LP.
Good luck!
Liusheng He, Ph.D, MD
Staff Scientist
Director, Flow Cytometry Facility
Office of Science and Technology
NIAMS, NIH
Bldg10, Rm6D52
9000 Rockville Pike
Tel (301)594-3531
Fax (301)402-2209
Lihe@mail.nih.gov
http://www.irp.niams.nih.gov/NIAMS2/LabsBranches_groups.jsp?branchId=63&grou
We use GFP and
YFP all the time on a Vantage with the 488 laser.Just
use a 510 (GFP) a 555 (YFP) and a 530 SP filter.
The problem is more interesting when
using CFP, GFP, YFP....but that can still be done easily with the right filters
and lasers.
Andy Johnson
FACS Facility Manager
Biomedical Research Centre
UBC
604-822-7838
Omega sells a filter set specifically for
this: Part number XCY-500. It consists of a 525SP dichroic, 510/21 and
550/30 bandpass filters (GFP and YFP respectively).
We have this set and have had good results
with it, provided the GFP and YFP signals are reasonably bright - but not *too*
bright, otherwise ND filters are needed. [ As an
aside, some of our investigators bring cells that are so bright you can see
them flash as they go through the laser beam!! ].
I have not had to retune the laser. Sorter is a Facstar
Plus.
Of course, I am assuming that by
"GFP" you don't mean wild type GFP; the latter is pretty dim and the
once or twice I encountered it, it was difficult to
separate the signal from autofluorescence.
- David Chambers, Salk Inst.
You're going to need a special filter
set, and then it should be no problem with a little fiddling with the FL1/FL2
dichroic. I've performed this analysis
on our old FACStar+ and now on our FACSVantageSE with great success.
The filter set we use is from Omega
Optical and includes a 510/20 for GFP, a 550/30 for YFP and a 530SP dichroic.
Gayle Thornbury
Terry Fox Lab
Thanks again to all:
