This document is a summary of message
from the
SNARF issues
Karim Y. Vermaelen,
MD, PhD
Cellular and Molecular Biology Research
Laboratory
Building 37 - Room 1096
NASA - Lyndon B.
2101 NASA Parkway
Houston, Texas 77058
tel (+281 24)4-1993
fax (+281 48)3-3058
Original
Question
Has anyone here some experience in loading
cells with SNARF-1? We need to label human dendritic
cells with this dye. I have a protocol that works very well for CFSE / CFDAse. As far as I understand, the labeling mechanism
should be similar, but I don't know whether the same concentration, incubation
times would apply to the SNARF-ester.Any suggestions
would be greatly appreciated!
------------------------------------------------------------------------------------------------------
Thanks to everyone who answered my
question on SNARF-1 labeling. A compilation of answers is provided
below. I ended up using exactly the same
protocol as for CFSE ( 20 x 10E6 DCs/ml, 5 microM SNARF-1 final conc., incubate 5 min at RT in HBSS, wash 3x
with serum-rich medium) -it worked
perfectly (intensity, viability).
Responses:
Dear Karim - I
have used Snarf before ... although it has been a
long time so my memory might be wrong. However, i'm
fairly certain i found that the viability with Snarf was similar to the CFSE concentration we were using (we got
these both from Molecular Probes).... so i label with
the CFSE/or/SNARF in PBS... at a range from aroudn
0.5uM to 2uM.. although in my experience, going higher causes cells (T
cells) to start dying...
hope that might help-
Mark
Hi Karim,
We have previously used SNARF-1 in
parallel with CFSE to stain PBMC and the staining methods were the same (10 mins at 37'C, quench staining with serum followed by
washes). You might want to test the toxicity of SNARF by reversing the dyes on
your stained populations to compare cell viability. CFSE is safe up to 5uM and
we've used SNARF at the same concentration and it seems okay. The intensity isn't
quite as intense but I wouldn't recommend anything above 5uM.
Good luck and cheers.
--
Socheata Chea
Department of Microbiology &
Immunology
The
Phone: 61 3 8344 9938
Fax: 61 3 8344 3846
Hi Karim,
First thing is to order the right product
- SNARF-1-AM, preferably 'special packaging' (Molecular Probes
C-1272). I follow largely the method of Chow and Hedley in 'Current
Protocols in Cytometry' (1997). Another good reference is Wieder et al Cytometry 14(8), 916-21, 1993. Old, but good for technique. Like them, I find time
and temp of labelling has little effect and use room
temp for 30 min (RT to avoid shift of pH with temp change on the FACS).
Buffers I would suggest to try are medium (no serum), PBS or HeBS (145mM NaCl, 5mM KCl, 1mM CaCl2, 0.5mM MgSO4, 5mM glucose, 10mM Hepes pH 7.4). I label with 5ug/ml of SNARF, the
concentration does seem to matter, if you don't have a red pellet they aren't labelled.
To read, we excite at 514nm to avoid GFP
interference but 488 is fine, and measure 640nm/580nm
emission. You need to do a calibration curve with each expt. I calibrate using the nigericin
method of Chow and Hedley.
Hope this answers your question, best of
luck.
Kellie Tainton
kellie.tainton@petermac.org
Hi Karim,
when you’re using SNARF®-1 carboxylic acid,
acetate, succinimidyl ester, the same conditions as
for CFDA-SE, i.e. 1.10 ug/ml in PBS and approx. 30
minutes, should give good results.
Hope this helps,
Arne
Hi, Karim:
We have somebody in the lab who had
success with labelling myoblast
cells and bone marrow SP cells with 20uM CFSE(from
Molecular Probe) (in Opti-MEM I medium without serum)
at 37C for an hour. I was able to detect the SNARF staining later using FACS.
Nan
Department of Internal Medicine
Cardiology Division
UT Southwestern Medical center at
Begin forwarded message:
> From: Karim
Vermaelen <kvermael@ems.jsc.nasa.gov>
> Date:
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: SNARF-1 labeling
>
> Hi,
