 |
 |
 |
 |
>>I have been doing flow on BAL (bronchoalveolar lavage) .... A problem
>>that seems to nag at us is that the Vantage, having a stream in air
>>setup, has only a 70 micron aperture and is subject to clogging even
>>though we filter it through 50 micron nylon mesh.
>>
.................................[edited]...............................
>>
>>My proposed solution is to spike each sample with non-fluorescent beads
>>of say 2-3 microns in diameter. My hope is that they show up as a sharp
>>peak just to the left of the lymphs. Movement of that peak would be
>>more obvious and would alert the operator that there was trouble.
>>
>>Questions for the group:
>>1.) Is this overkill?
>>2.) Are there other simpler or better ways to do this?
>>3.) Will it work at described above? What size beads should I use?
>
-------------------------------Reply-----------------------------------
I suspect your bronchoalveolar lavage cells like our intestinal epithelial
lymphocytes (I EL's)are producing mucin, particularly when heat stressed
and/or during handling. Although filtering through 50 micron mesh was
performed immediately prior to sorting, clogging would occur after a period
of time.
We did the following to reduce mucin production and eliminate the problem
of clogs caused by it. Cells were stained and fixed with paraformaldehyde
in the normal fashion, then washed to remove any paraformaldehyde.
Hyaluronidase was added to the cell pellet and PBS added to resuspended
them. Cells were passed through a 50 micon nylon mesh and then placed on
the FACS Vantage. Gentl aggitation was used to resuspend the cells during
long sorts.
We found that by maintaining the sample aliquot at 5 degrees while sorting,
little or no mucin was being produced to the point where it would start
forming clogs in the 70 micon nozzle. We have a very tight gate set around
the IEL cells and no drifting is observed over long sorts. To monitor the
condition of the nozzle, I use Polysciences 2.02 micron YG beads before and
after the sort, occasionally between sample changes to record the C.V.'s of
the FSC, SSC and FL-1/Fl-2 peaks. Values have remained the same since doing
this QC check.
To clean the system and nozzle, I run a tube of hyaluronidase through the
sample uptake tubing followed by a water rinse. 0.5% bleach is then run
followed by a sterile water rinse. The nozzle is removed and placed into
hyaluronidase and soaked at 37 c, then sonicated for 15-30 seconds. Water
is forced backward through the nozzle tip, then reattched to the sorter.
These procedures have proven to be effective in doing long sorts without
mucin causing the cells to clump and clog the nozzle.
Hyaluronidase is purchased from Sigma, Cat Nr. H2126, Type II from sheep.
An alternate enzyme is muramidase (lysozyme) also supplied by Sigma.
Steven Merlin
University of Bern
FACS Core Facility
Institute for Immunology and Allergy
Murtenstrasse 31
CH-3010 Bern
Switzerland
Voice: ++41-31-632-8876
FAX: ++41-31-381-8764
e-mail: merlin@patho.unibe.ch
|
|
|
|