minimacs/FACS final

Carolyn Jefferiss prscmj at bath.ac.uk
Mon Aug 21 06:27:41 EST 2000


Dear Everyone,

Does anyone put DNase 1 in their sort sample, to help reduce
clumping, to good effect?

ALSO:-
I enclose the answers to my previous questions for anyone else who is
interested,as attachments, as some went out to the List and some came
directly to me; and a brief summary below.

  And, I apologise if my questions seemed ambiguous. It proved
hazardous to discuss "sorting" by two different methods!
  In retrospect it  would have been clearer to refer to "isolating" on
the minimacs and "sorting" by flow cytometry.

I expect everyone would like a preliminary step to reduce the
negative population so that Flow Cytometric sorting can be performed
more quickly, by getting rid of the  bulk of irrelevant cells, and
this is one of the things  I am trying to do. Asking these questions
is me trying to save on finding the answers empyrically because beads
are so horrendously expensive.

  1) I intended to find out if the magnetic nature of the initial
separation by minimacs would leave magnetic particles on the cells
which would cause them to be attracted to the difflection plates on
the flow cytometer.

  Others have done this and find no interference from the beads,
although 'bead manufacturers' have intimated that if the cytometer
has any magnetic valves ( apparently some do) beads may accumulate
here.

2)Possible  "loss" of label; I haven't been re-labelling the
POSITIVELY selected cells with primary antibody; just adding
conjugated secondary for flow cytometric analysis of purity. e.g.
anti-glycophorinA coated beads  for minimacsing, then anti-IgGFITC
conjugate for FACS checking.

The general expectation of purity obtained with magnetic beads from a
magnetic column seems to be lower than sorting - that is, happy with
70-80% whereas I was hoping for something nearer the 95+% I've had
with Flow cytometry. What puzzled me was how the unlabelled cells
were present, given that they weren't magnetically bound to the
column.
Dead cells binding  non-specifically to beads was one answer. Amount
of antigen present on the cells  was another. However, as others
regularly obtained 90-99% positivity it is probably my technique
which is reducing purity.

  (I follow the manufacturer's recommended wash procedure, but still
don't know  how much more I can wash the column.)

3) Sorting time:  A useful ref. given; " Cytometry 12:268-274 (1991)"
	Also advice;
	for rare cells	- use fluorescence trigger 'enrich' mode and
a high sort rate; followed by a resort with scatter trigger, in
'Normal' mode and a more dilute sample.





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Hi Carolyn

> 1) Magnetic beads and Flow Sorting:
> I have presumed that the beds (Miltenyi) would interfere
> with sorting by flow cytometry, even though they are
> absolutely titchy. Please say if you have sorted cells from
> a population containing magnetically-labelled cells, to
> good effect.

We have taken Cd34 (indirect antibody)column selected cells
from BM and then sorted by direct labelling with a PE
conjugate against a different epitope and with labels for
other targets eg.CD59. Top results, managed to get clean
sub-fraction of CD34 pos population

> 2) Magnetically labelled cells:
> When checking magnetically labelled and isolated cells for
> purity by flow cytometry, has anyone looked at loss of
> label? I was wondering whether or not there is a
> significant loss of label somewhere from the positive
> fraction in coming off the column or later. - I
> do everything at 4degrees and despite using the usual
> concentrations of conjugated second abs see a significant
> proportion of totally unlabelled cells in the fraction
> which was positively bound to the magnetic column.

Try staining for column on ice, although we get good
results (90-99% pure) at 4-10 degC (temp.regulated fridge,
min-max thermometer very useful) for 15min for each layer
of antibody.

> 3)Sorting time:
> Finally, When sorting a rare population from whole bone
> marrow, what kind of sort rate should be used? How stable
> could I expect "my" Vantage to be over the number of hours
> I'd need to use it? I am extremely impressed by the
> indication that other users seem to have a stable settup
> for many hours. I have never sorted for more than
> four hours. I'd need to run at least 100 million cells to
> get enough of my chosen population.

We have a FacStarPlus. We do fast enrich (if not column
enriched), followed by slow dilute sort, we've had the
thing going for 8hours while remaining stable, however we
do have an environmentally controlled environment


Good luck!!
-----------------------------------
sian rizzo
@sghms.ac.uk
St. Georges Hospital Medical School
Opinions expressed those of the author and not the institution

-------------- next part --------------
Hi
I have no experience in flow sortiing of magnetically sorted cells. However
in the past I have isolated B cells using CD 19 coated beads from Miltenyi.
Following sorting, I stained the cells with anti cd 19-FITC and I got over
98% positivity. so I used the same ligand for both sorting and analysis.  A
friend of mine told me he always leaves the cells in tissue culture medium
overnight for the beads to dissociate from the cells however, I did not
encounter this problem.

Please note that we do the magnetic sorting at room temperature and always
the cells sit for a while(45 min to 2 hrs) in tissue culture medium before
we stain them.

Mutasim
-----Original Message-----
From: Carolyn Jefferiss <prscmj at bath.ac.uk>
To: cyto-inbox
Date: Tuesday, August 08, 2000 2:04 PM
Subject: magnetic beads and/or sorting


>
>Dear Everyone,
>Three things:-
>1) Magnetic beads and Flow Sorting:
>I have presumed that the beds (Miltenyi) would interfere with sorting
>by flow cytometry, even though they are absolutely titchy. Please say
>if you have sorted cells from a population containing
>magnetically-labelled cells, to good effect.
>
>2) Magnetically labelled cells:
>When checking magnetically labelled and isolated cells for purity by
>flow cytometry, has anyone looked at loss of label? I was wondering
>whether or not there is a significant loss of label somewhere from
>the positive fraction in coming off the column or later. - I do
>everything at 4degrees and despite using the usual concentrations of
>conjugated second abs see a significant proportion of totally
>unlabelled cells in the fraction which was positively bound to the
>magnetic column.
>
>3)Sorting time:
>Finally, When sorting a rare population from whole bone marrow, what
>kind of sort rate should be used? How stable could I expect "my"
>Vantage to be over the number of hours I'd need to use it? I am
>extremely impressed by the indication that other users seem to have a
>stable settup for many hours. I have never sorted for more than four
>hours. I'd need to run at least 100 million cells to get enough of my
>chosen population.
>
>
>Thank you for your attention.
>Carolyn Jefferiss
>Carolyn Jefferiss Ph. D.
>Pharmacy and Pharmacology
>University of Bath
>Claverton Down
>Bath BA2 7HY
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1) They don't matter. We've also use the GIANT Dynal beads, and the
contaminating beads are vastly different from unlabeled cells. You can even
see bead:cell aggregates.

2) Most use a different labeling epitope or, if lucky, not all epitopes are
used in either labeling step (magnetic or fluorescent).

3) Check the original Herzenberg articles (They sorted whole BM in 9 hours
with antique hardware!), or a more recent one: McCoy, Chambers, Lakomy,
Campbell & Stewart, Sorting of Minor Subpopulations of Cells: Use of
Fluorescence as the Triggering Signal, Cytometry 12:268-274(1991). Remember
that FACS means Fluorescence-activated cell sorting.
	a) Concentrate sample
	b) Set trigger to fluorescence and sort in enrich mode. Since you can sort
a 1% pop at 8,000 per second, you actually process 800,000 per second.
	c) Concentrate and re-sort with scatter trigger in Normal mode.
(see


At 09:35 AM 8/8/00 +0100, you wrote:
>
>Dear Everyone,
>Three things:-
>1) Magnetic beads and Flow Sorting:
>I have presumed that the beds (Miltenyi) would interfere with sorting
>by flow cytometry, even though they are absolutely titchy. Please say
>if you have sorted cells from a population containing
>magnetically-labelled cells, to good effect.
>
>2) Magnetically labelled cells:
>When checking magnetically labelled and isolated cells for purity by
>flow cytometry, has anyone looked at loss of label? I was wondering
>whether or not there is a significant loss of label somewhere from
>the positive fraction in coming off the column or later. - I do
>everything at 4degrees and despite using the usual concentrations of
>conjugated second abs see a significant proportion of totally
>unlabelled cells in the fraction which was positively bound to the
>magnetic column.
>
>3)Sorting time:
>Finally, When sorting a rare population from whole bone marrow, what
>kind of sort rate should be used? How stable could I expect "my"
>Vantage to be over the number of hours I'd need to use it? I am
>extremely impressed by the indication that other users seem to have a
>stable settup for many hours. I have never sorted for more than four
>hours. I'd need to run at least 100 million cells to get enough of my
>chosen population.
>
>
>Thank you for your attention.
>Carolyn Jefferiss
>Carolyn Jefferiss Ph. D.
>Pharmacy and Pharmacology
>University of Bath
>Claverton Down
>Bath BA2 7HY

---Dennis

Dennis J. Young
Flow Cytometry Core Facility
University of California, San Diego
Internal Medicine Group, Bldg #4, Rm 126
9500 Gilman Drive
La Jolla  CA  92093-0671

Mail:<<mailto:djyoung at ucsd.edu>>
WWW:<<http://cancer.ucsd.edu/SResources/flow.htm>>
Telephone:(858) 822-0407	FAX: (858) 822-0403
-------------- next part --------------
Dear Carolyn
We have FACS sorted previously magnetic bead (MACS) labelled cells with good effect, in
fact it is a technique which we make a lot of use of. You don't say what you are
labelling for but we find that the "efficiency" of the MACS sort largely depends on the
antigen, broadly speaking if there is a lot of antigen (say CD4 on thymocytes) then the
technique works well, 80-90% cells you want. However if the antigen has lower
expression (say CD34 in bone marrow) it does not select the cells as well say 60%
purity. As far as the sorter is concerned if you get no blockages and the nozzle is
nice and clean etc it should remain stable all day (not withstanding a bad blockage 15
mins from the end of the sort!!! sample preparation IS important - no clumps)

Hope this is helpful

Ian

Ian Titley PhD
Leukaemia Research Fund Centre
at the Institute of Cancer Research
237 Fulham Road
LONDON SW3 6JB UK
Tel Direct: +44 (0)20 7970 6048
or Tel: +44 (0)20 7352 8133 ext5134
Fax: +44 (0)20 7352 3299
E-mail: iant at icr.ac.uk

On Tue, 8 Aug 2000 09:35:56 +0100 Carolyn Jefferiss <prscmj at bath.ac.uk> wrote:

>
> Dear Everyone,
> Three things:-
> 1) Magnetic beads and Flow Sorting:
> I have presumed that the beds (Miltenyi) would interfere with sorting
> by flow cytometry, even though they are absolutely titchy. Please say
> if you have sorted cells from a population containing
> magnetically-labelled cells, to good effect.
>
> 2) Magnetically labelled cells:
> When checking magnetically labelled and isolated cells for purity by
> flow cytometry, has anyone looked at loss of label? I was wondering
> whether or not there is a significant loss of label somewhere from
> the positive fraction in coming off the column or later. - I do
> everything at 4degrees and despite using the usual concentrations of
> conjugated second abs see a significant proportion of totally
> unlabelled cells in the fraction which was positively bound to the
> magnetic column.
>
> 3)Sorting time:
> Finally, When sorting a rare population from whole bone marrow, what
> kind of sort rate should be used? How stable could I expect "my"
> Vantage to be over the number of hours I'd need to use it? I am
> extremely impressed by the indication that other users seem to have a
> stable settup for many hours. I have never sorted for more than four
> hours. I'd need to run at least 100 million cells to get enough of my
> chosen population.
>
>
> Thank you for your attention.
> Carolyn Jefferiss
> Carolyn Jefferiss Ph. D.
> Pharmacy and Pharmacology
> University of Bath
> Claverton Down
> Bath BA2 7HY



-------------- next part --------------

Hi

1. The MACS beads are indeed tiny and do not interfere with sorting at all.

2. If you are checking the purity of your cells with an antibody recognizing the same
epitope as the MACS beads, you will have problems as many or most of the epitopes
will already be occupied with the antibodies attached to the beads. You need another
antibody which does not compete with the first one.

3. My Vantage is stable over many hours with only minor adjustments of the amplitude knob
to keep the phase and break-off in the same place. You may have to back-flush and perhaps
do a nozzle flush every now and then to clear gunk from the nozzle depending on sample
prep. If you are sorting rare cells one trick is to increase the sample rate up to about
the same as the drop drive frequency, change the theshold to the fluorescent channel
which you have the rare cell marker in, increase the theshold until all the negative
cells disappear and change sort mode to enrich. If your original sample rate was 27K
(with no turbo sort and 70u noz) and your population is 1% then after increasing the
theshold the new sample rate will be about 270. With a three drop envelope your purity
will be in the region of 33% if you sort all cells above theshold. You then spin then
down and resort using normal theshold, rate and normal-R sort mode. When doing your
enrichment sort you should make your sort windows generous, scatter will not be that
tight. You should also make sure you have no lumps in your samples, hanging a filter
off your sample probe is best.

Good luck



Simon Monard
FACS Lab Manager
Trudeau Institute
Saranac Lake
NY12983

Ph 518 891 3080 X352


>>> Carolyn Jefferiss <prscmj at bath.ac.uk> - 8/8/2000 4:35 AM >>>

Dear Everyone,
Three things:-
1) Magnetic beads and Flow Sorting:
I have presumed that the beds (Miltenyi) would interfere with sorting
by flow cytometry, even though they are absolutely titchy. Please say
if you have sorted cells from a population containing
magnetically-labelled cells, to good effect.

2) Magnetically labelled cells:
When checking magnetically labelled and isolated cells for purity by
flow cytometry, has anyone looked at loss of label? I was wondering
whether or not there is a significant loss of label somewhere from
the positive fraction in coming off the column or later. - I do
everything at 4degrees and despite using the usual concentrations of
conjugated second abs see a significant proportion of totally
unlabelled cells in the fraction which was positively bound to the
magnetic column.

3)Sorting time:
Finally, When sorting a rare population from whole bone marrow, what
kind of sort rate should be used? How stable could I expect "my"
Vantage to be over the number of hours I'd need to use it? I am
extremely impressed by the indication that other users seem to have a
stable settup for many hours. I have never sorted for more than four
hours. I'd need to run at least 100 million cells to get enough of my
chosen population.


Thank you for your attention.
Carolyn Jefferiss
Carolyn Jefferiss Ph. D.
Pharmacy and Pharmacology
University of Bath
Claverton Down
Bath BA2 7HY
-------------- next part --------------
Hi, below are my thought on that

>
> Dear Everyone,
> Three things:-
> 1) Magnetic beads and Flow Sorting:
> I have presumed that the beds (Miltenyi) would interfere with sorting
> by flow cytometry, even though they are absolutely titchy. Please say
> if you have sorted cells from a population containing
> magnetically-labelled cells, to good effect.

I does work, we did it often.

> 2) Magnetically labelled cells:
> When checking magnetically labelled and isolated cells for purity by
> flow cytometry, has anyone looked at loss of label? I was wondering
> whether or not there is a significant loss of label somewhere from
> the positive fraction in coming off the column or later. - I do
> everything at 4degrees and despite using the usual concentrations of
> conjugated second abs see a significant proportion of totally
> unlabelled cells in the fraction which was positively bound to the
> magnetic column.

There are two issues here: 1) you get dead cells which unspecifically bind
microbeads and can give you funny results in flow; 2) you have to be careful
about the choice of your antibodies, if the see the same epitope/compete you
have a problem

>
> 3)Sorting time:
> Finally, When sorting a rare population from whole bone marrow, what
> kind of sort rate should be used? How stable could I expect "my"
> Vantage to be over the number of hours I'd need to use it? I am
> extremely impressed by the indication that other users seem to have a
> stable settup for many hours. I have never sorted for more than four
> hours. I'd need to run at least 100 million cells to get enough of my
> chosen population.

 Sorry, not enough experience with that

>
> Thank you for your attention.
> Carolyn Jefferiss
> Carolyn Jefferiss Ph. D.
> Pharmacy and Pharmacology
> University of Bath
> Claverton Down
> Bath BA2 7HY
>

Jan Muller-Berghaus, M.D.
Research Associate
Department of Surgery
University of Pittsburgh
200 Lothrop St
Biomedical Science Tower, Room W1505
Pittsburgh, PA 15261
T# 412-383-7653
F# 412-624-1172
E# muellerberghausj at msx.upmc.edu
-------------- next part --------------

Carolyn,

My experiences from my previous job:

1)  We successfully sorted pre-enriched populations (using the Miltenyi
MACS system) with no interference evident.

2)  We did not notice a significant diminution of signal when following
the purity of MACS enriched populations.  We did, however, see a
variation in purity depending on the source of cells and the type of
purification (positive versus negative selection).  By counterstaining
the populations, we were able to account for most of the contaminating
cells.

3)  We did sort rare populations from mouse spleen using a FACStar plus,
but I cannot remember the rate.  It did take a long time to get a
significant number of cells, but we were looking at about a 1% maximum
population.

Randy Fischer
TherImmune Research Corporation
9700 Great Seneca Hwy
Rockville, MD 20850
(240) 453-6256
RFischer at therimmune.com

> ----------
> From:		Carolyn Jefferiss
> Sent:		Tuesday, August 8, 2000 1:35 AM
> To:	Cytometry Mailing List
> Subject:	magnetic beads and/or sorting
>
>
> Dear Everyone,
> Three things:-
> 1) Magnetic beads and Flow Sorting:
> I have presumed that the beds (Miltenyi) would interfere with sorting
> by flow cytometry, even though they are absolutely titchy. Please say
> if you have sorted cells from a population containing
> magnetically-labelled cells, to good effect.
>
> 2) Magnetically labelled cells:
> When checking magnetically labelled and isolated cells for purity by
> flow cytometry, has anyone looked at loss of label? I was wondering
> whether or not there is a significant loss of label somewhere from
> the positive fraction in coming off the column or later. - I do
> everything at 4degrees and despite using the usual concentrations of
> conjugated second abs see a significant proportion of totally
> unlabelled cells in the fraction which was positively bound to the
> magnetic column.
>
> 3)Sorting time:
> Finally, When sorting a rare population from whole bone marrow, what
> kind of sort rate should be used? How stable could I expect "my"
> Vantage to be over the number of hours I'd need to use it? I am
> extremely impressed by the indication that other users seem to have a
> stable settup for many hours. I have never sorted for more than four
> hours. I'd need to run at least 100 million cells to get enough of my
> chosen population.
>
>
> Thank you for your attention.
> Carolyn Jefferiss
> Carolyn Jefferiss Ph. D.
> Pharmacy and Pharmacology
> University of Bath
> Claverton Down
> Bath BA2 7HY
>
>
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Well to Number 1

I ran this by one of the bead mfgs and they were against putting magnetic
beads in a cytometer. they said some have magnetic valves and would cause
beads to accumulate etc. I have a BDIS FacStar Plus and I have run mag beads
in it before and did not percieve a problem. So I don't know if the company
was right or not.

I have not sorted with beads via the cytomeeter though. We were using the
beads as a non centrifuged preperation with our own
target attatched to the beads

Good luck

Pb

-----Original Message-----
From: Carolyn Jefferiss [mailto:prscmj at bath.ac.uk]
Sent: Tuesday, August 08, 2000 4:36 AM
To: cyto-inbox
Subject: magnetic beads and/or sorting



Dear Everyone,
Three things:-
1) Magnetic beads and Flow Sorting:
I have presumed that the beds (Miltenyi) would interfere with sorting
by flow cytometry, even though they are absolutely titchy. Please say
if you have sorted cells from a population containing
magnetically-labelled cells, to good effect.

2) Magnetically labelled cells:
When checking magnetically labelled and isolated cells for purity by
flow cytometry, has anyone looked at loss of label? I was wondering
whether or not there is a significant loss of label somewhere from
the positive fraction in coming off the column or later. - I do
everything at 4degrees and despite using the usual concentrations of
conjugated second abs see a significant proportion of totally
unlabelled cells in the fraction which was positively bound to the
magnetic column.

3)Sorting time:
Finally, When sorting a rare population from whole bone marrow, what
kind of sort rate should be used? How stable could I expect "my"
Vantage to be over the number of hours I'd need to use it? I am
extremely impressed by the indication that other users seem to have a
stable settup for many hours. I have never sorted for more than four
hours. I'd need to run at least 100 million cells to get enough of my
chosen population.


Thank you for your attention.
Carolyn Jefferiss
Carolyn Jefferiss Ph. D.
Pharmacy and Pharmacology
University of Bath
Claverton Down
Bath BA2 7HY
-------------- next part --------------
Carolyn Jefferiss Ph. D.
Pharmacy and Pharmacology
University of Bath
Claverton Down
Bath BA2 7HY


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