Howard Shapiro writes:
>Ron Rabin writes:
>
>>I am planning to do 4 color analysis + cell cycle on a dual laser Vantage
>>that can use either a dye laser or a UV laser as the second laser. I can
>>use either FITC, PE, PECy5, Red613 and Hoechst, or I can use FITC, PE, 7ADD,
>>R613, and APC. Because my PE conjugates are critical to the experiment, I
>>cannot use the Pyronin Y. It seems the Hoechst would be easier, cell
>>permeable etc, though I have no experience with it.
>>
>
>If you have no problems resolving Red613 from PE and PECy5, doing the
>4-color immunofluorescence in the 488 beam and Hoechst 33342 in the UV beam
>is probably preferable to trying to get a good DNA histogram with 7-AAD.
>Whether or not you can get good staining without fixing or permeabilizing
>the cells depends on cell type; incubation at 37 C with 3-5 ug/ml Hoechst
>33342 for 30 minutes or so generally gives good staining of viable human or
>rat lymphoid cells, but many strains of mice seem to pump out the dye, and
>some hematopoietic cells, e.g., the more primitive CD34+ cells, from many
>mammalian species are Hoechst-dim.
>
>At 1 ug/ml, Hoechst 33342 gives good DNA histograms in fixed cells,
>particularly when 0.1% Triton X-100 is added. However, I (and others) have
>observed, but not formally published, that staining of formaldehyde-fixed
>cells with Hoechst dyes and DAPI produces increases in fluorescence
>background in the fluorescein and PE channels. We don't understand the
>mechanism, but believe that the fluorescence is due to a chemical reaction
>between the dye and formaldehyde or some product of formaldehyde. The
>longer the time in fixative, the worse the background; once cells have been
>adequately fixed, adding 0.15% glycine (a suggestion made to us by Richard
>Riese) seems to neutralize the remaining fix and prevent further increases
>in background fluorescence. If I weren't trying to use the formaldehyde to
>neutralize HIV and other nasties in specimens, I'd be tempted to work with
>unfixed or ethanol-fixed cells.
>
>-Howard
A FACS Vantage SE can measure 4 fluorochromes simultaneously excited by one
laser (488nm exciting FITC, PE, PE-TR and [PE-Cy5 or 7-AAD] in this
example); a regular FACS Vantage can NOT (it can measure only 3). However,
there was an article recently published that described using 488nm
excitation from BOTH the first and second lasers on a regular FACS Vantage
to measure more than 3 488nm-excited fluorochromes simultaneously. Unless
you have OmniComp or software compensation, however, you will not be able
to compensate between the first laser signals (FL1, FL2, FL3) and the
second laser signals (FL4, FL5). This limitation can be a problem.
In order to do 5 colors on a Vantage, you need to excite 3 dyes with your
first laser and 2 dyes with your second (and possibly third) laser(s).
Since it would be difficult to excite a 2nd dye along with a Hoechst or
DAPI dye using 350-365nm UV excitation - and due to the "488nm-excitable"
nature of these dyes noted by Howard Shapiro above (we've seen this too) -
I would suggest using the dye laser as your second laser. The dye laser can
be tuned to 630nm* or higher to excite APC and APC-Cy7** (you may need a
red-sensitive PMT to detect APC-Cy7, as Mario Roederer has pointed out here
before). Now you still need a DNA dye. Molecular Probes
<http://www.probes.com/> makes several DNA dyes. Perhaps you could use
SYTOX Green or YOYO-1; each is excited with 488nm and fluoresces "green"
similar to fluoroscein, with SYTOX Green more red-shifted (i.e., more
spectral overlap with PE) than YOYO-1. The remaining two fluorochromes
could be PE and PE-TR*** (Red613 or ECD).
*You may want to optimize the wavelength of the dye laser to maximize APC
fluorescence while minimizing PE-TR fluorescence.
**You could use Texas Red (and tune the dye laser accordingly) instead of
APC-Cy7, but then you'd have to find a way to compensate between TR and
PE-TR. Alternatively, you could use PE-Cy7 (and tune the dye laser
accordingly) instead of PE-TR, but then you still need a red-sensitive PMT.
***You could try using PE-Cy5 instead of PE-TR, but then you'd have to find
a way to compensate between PE-Cy5 and APC (or use PE-Cy7 instead of PE-TR).
In summary, I would suggest either of the following two setups (although I
haven't tried them myself):
(1) YOYO-1, PE, PE-TR, APC, APC-Cy7
(2) YOYO-1, PE, PE-Cy7, TR, APC
Anybody have any thoughts/experience with these setups?
If you could run all 3 lasers (488nm, UV and dye) simultaneously, that
could open up even more possibilities.
For more information about compensation values when using some of these
fluorochromes with 488nm and 595nm lasers, see
8 Color, 10-Parameter Flow Cytometry to Elucidate Complex Leukocyte
Heterogeneity. Mario Roederer, et. al. Cytometry 29:328-339 (1997).
or maybe even Mario Roederer's site at <http://www.drmr.com/>.
Although not as promising, you could also try exciting Cascade Blue and
Cascade Yellow with your UV laser. This was done with the 407nm line from a
krypton laser in
Pairs of Violet-Light-Excited Fluorochromes for Flow Cytometric
Analysis. M.T. Anderson, et. al. Cytometry 33:435-444 (1998).
By the way, another problem with 7-AAD is that it would be excited with the
dye laser. The Hospital for Special Surgery (HSS) describes a 4 color setup
(DNA plus 3 colors) on the Calibur using either 7-AAD or LDS 751 for the
DNA dye at <http://hss.hss.edu/research/flow/hssflow.htm>. LDS 751 may be a
better choice than 7-AAD (FITC, PE, LDS 751, TR and APC, for example). The
same site has information about OmniComp.
Good luck,
Eric
/\/\/\_ Eric Van Buren, aa9080@wayne.edu
\ \ \ Karmanos Cancer Institute and Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan, USA
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