Choosing a Nozzle
Our IDDRC Stem Cell Flow
Cytometry Core has three nozzle sizes, a 70 micron nozzle, an 85 micron nozzle
and a 100 micron nozzle.
Important things to
consider in choosing a nozzle size for your sample and experiment:
1. Size of the largest cells in your sample
population
2. Sample concentration and sample volume to sort
3. Number of target cells expected/ desired from
the sort
4. Fragility of the target cells
Some Pros and Cons of
each nozzle type
70 micron Nozzle with cell concentration greater than 10 million cells/ ml
1. Very useful for
sorting small cells, (< 25 u) from a large volume with a high
concentration of cells
2. Cell prep needs to be
very clean, i.e., free from sticky cell debris and clumped cells
3. Nozzle clogs
relatively easily- small opening- must bring your 20 u, 25 u or 35 u strainers
and pass your samples through the strainer at Cell Sorter Core facility just
before beginning sorting process if cells tend to clump
4. Can process 10,000
events/ sec
5. Ideal concentration
is approximately 10 million cells/ ml
6. Can sort 40 million
sample cells with a sample cell concentration of approximately10 million
cells/ml in 67 minutes after sort set up if there are no clogs.
100 micron Nozzle with cell concentration of less than 4 million cells/ ml
1. Best choice for
fragile cells
2. Can sort larger
cells, up to 40u
3. Nozzle is very
stable, resists clogging.
4. Can process 3700
events/ sec
5. Best choice for
single cell sorting
6. Best choice for
sorting small percentage cell populations (<1%)
7. Ideal concentration
approximately 3.7 million cells/ ml
8. Can sort 15 million
sample cells with a sample cell concentration of approximately 3.7million
cells/ml in 67 minutes after sort set up if there are no clogs.
Cells in suspension must
be passed through a strainer to remove clumps immediately before sorting by
FACS. Using strainers immediately before analysis or sorting is one way to
ensure that cells are below a certain size and not clumped together.
Some available strainers
include:
1] EMD Millipore
Corporation
290 Concord Road
Billerica MA 01821
800-645-5476 or
978-715-4321
Steriflips 20 u and 40 u
25/pkg = $147.29
2] BD Falcon/BD
Biosciences 35 u cell strainers available from VWR at:
https://us.vwr.com/store/catalog/product.jsp?catalog_number=21008-948
Case of 500= $651
3] BD Falcon/BD
Biosciences cell strainers available from VWR at: https://us.vwr.com/store/catalog/product.jsp?catalog_number=21008-949
Case of 50 = $135.42
Unfortunately, nothing
smaller than 40 u is currently offered.
4]Fisherbrand Cell
Strainers
Cat. # cell strainer
size Price/Case of 50
22-363-547 40um, blue
$100.50
22-363-548 70um, white
$100.50
22-363-549 100um, yellow
$100.50
5] BioDesign Inc. of New
York
BioDesign
CellMicroSieves
Pore Size (microns)
Sheets (30x30 cm), 3/pkg Roll(61 x 91.5 cm) 47mmCircles/10
200u N200S $63.00 N200R
$109.00 N200C $35.00
100u N100S $64.00 N100R
$111.00 N100C
70u N70S $65.00 N70R
$113.00 N70C
50u N50S $67.00 N50R
$116.00 N50C
35u N35S $69.00 N35R
$121.00 N35C
25u N25S $71.00 N25R
$124.00 N25C
20u N20S $73.00 N20R
$133.00 N20C
15u N15S $75.00 N15R
$136.00 N15C
10u N10S $81.00 N10R
$149.00 N10C
8u N8S $88.00 N8R
$162.00 N8C
5u N5S $89.00 N5R
$164.00 N5C
Also have sample packs
made up of 12”x12” square sheets of 5 pore size selections.
Cell size can be
estimated under a microscope.
Use an eyepiece reticule
(aka: eyepiece micrometer, eyepiece reticule, eyepiece graticule) such as:
Part number: KR 207
Klarmann Rulings, Inc.
480 Charles Bancroft
Highway
Litchield, NH 03052
800-252-2401
Eyepiece reticules must
be calibrated before each use or at least before each change of objective lens
using a stage micrometer (aka: stage graticule) such as:
Part number: 2285-15
horizontal scale only $434.25
or 2285-16 crossed
scales $420.50 from
Ted Pella, Inc.
P.O. Box 492477
Redding, CA 96049-2477
800-237-3526
Another useful tool is
the handheld (pipette like) cell counter from EMD Millipore called the
“Scepter”. It displays a histogram of cell diameters as well as concentration.
The human eye with 20/20
vision can discriminate/see a 100 u diameter particle.
For cells that average
20 u in diameter, a 40X objective is sufficient (i.e. equivalent of 800 u
particle size). A KR207 eyepiece micrometer is 100 u per division (and has 100
divisions) at 1X objective and will therefore magnify to 2.5 u per division at
40X. 10 divisions will cover a 25 u diameter cell.
