STEMCELL SepMate 50 Manual

FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
TOLL FREE PHONE [phone] PHONE [phone] VERSION 2.0.0
[email protected] TECHSUP[email protected] FOR GLOBAL CONTACT DETAILS VISIT WWW.STEMCELL.COM DOCUMENT #29251
INTENDED USE
SepMate™ tubes are designed for the in vitro isolation of
mononuclear cells (MNCs) from human whole peripheral blood and
cord blood samples by density gradient centrifugation.
PRODUCT DESCRIPTION
Polypropylene tube with insert. Gamma-irradiated.
STORAGE
Store at room temperature (15 - 25°C).
DIRECTIONS FOR USE
Ensure that sample, phosphate-buffered saline with 2% fetal bovine
serum (PBS + 2% FBS; Catalog #07905), density gradient medium
(see Notes on reverse page), and centrifuge are all at room
temperature (15 - 25°C).
1. Add density gradient medium to the SepMate™ tube by
carefully pipetting it through the central hole of the SepMate™
insert. Refer to Table 1 for required volumes. The top of the
density gradient medium will be above the insert.
Note: Small bubbles may be present in the density gradient
medium after pipetting. This will not affect performance.
2. Dilute sample with an equal volume of PBS + 2% FBS.
Mix gently.
For example, dilute 5 mL of sample with 5 mL of PBS +
2% FBS.
3. Keeping the SepMate™ tube vertical, add the diluted sample
by pipetting it down the side of the tube. The sample will mix
with the density gradient medium above the insert.
Note: The sample can be poured down the side of the tube.
Take care not to pour the diluted sample directly through the
central hole.
4. Centrifuge at 1200 x g(see Notes) for 10 minutes at room
temperature, with the brake on.
Note: For samples older than 24 hours, a centrifugation time of
20 minutes is recommended.
5. Pour off the top layer, which contains the enriched MNCs, into
a new tube. Do not hold the SepMate™ tube in the inverted
position for longer than 2 seconds.
Note: Some red blood cells (RBCs) may be present on the
surface of the SepMate™ insert after centrifugation. This will
not affect performance.
6. Wash enriched MNCs with PBS + 2% FBS. Repeat wash.
Note: Centrifuging at 300 x g for 8 minutes at room
temperature, with the brake on, is recommended.
SEPMATE™ PROCEDURE
Numbers in brackets refer to steps under Directions for Use.
Table 1: Density Gradient Medium Volumes
SEPMATE™
TUBE INITIAL BLOOD SAMPLE (mL) DENSITY GRADIENT MEDIUM (mL)
15 0.5 - 4.0 4.5
15 > 4 - 5 3.5
50 4 - 17 15
CATALOG #15415 20 TUBES/BAG
CATALOG #15420 5X20 TUBES/BAG
SepMate™-15
Centrifuge at
1200 xgfor 10 min
(4)
Add density
gradient medium
(1)
Pipette diluted sample
down the side of the tube
(3)
Wash enriched
MNCs with
PBS + 2% FBS.
Repeat wash
(6)
Pour
(5)
MNCs
Plasma
RBCs
Density gradient
medium
MNCs
SepMate™-50
CATALOG #15450 20 TUBES/BAG
CATALOG #15460 5X20 TUBES/BAG

Copyright © 2013 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, SepMate, and
RosetteSep are trademarks of STEMCELL Technologies Inc. Lymphoprep is a trademark of AXIS-SHIELD. Ficoll-Paque PLUS is a trademark of GE Healthcare Limited. All other trademarks are the property of their
respective holders.
FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
TOLL FREE PHONE [phone] PHONE [phone] VERSION 2.0.0
[email protected] TECHSUP[email protected] FOR GLOBAL CONTACT DETAILS VISIT WWW.STEMCELL.COM DOCUMENT #29251
CATALOG #15450 50 mL tubes, 20 tubes/bag
CATALOG #15460 50 mL tubes, 5 x 20 tubes/bag
CATALOG #15415 15 mL tubes, 20 tubes/bag
CATALOG #15420 15 mL tubes, 5 x 20 tubes/ba
g
NOTES
Samples
SepMate™ can be used with human whole peripheral blood and
cord blood samples. It has not been tested with samples older than
48 hours. For use of SepMate™ with samples other than human
whole peripheral blood or cord blood please contact STEMCELL
Technologies’ Technical Support at [email protected].
SepMate™-15
SepMate™-15 is designed to process 0.5 - 5 mL of initial sample.
A minimum packed RBC volume of 0.25 mL is required. For patient
samples with low hematocrits, the minimum sample volume may
therefore be greater than 0.5 mL.
There is a maximum packed RBC volume of 3 mL. For patient
samples with very high hematocrits, the maximum sample volume
may therefore be less than 5 mL.
SepMate™-50
SepMate™-50 is designed to process 4 - 17 mL of initial sample.
A minimum packed RBC volume of 2 mL is required. For patient
samples with low hematocrits, the minimum sample volume may
therefore be greater than 4 mL.
There is a maximum packed RBC volume of 12 mL. For patient
samples with very high hematocrits, the maximum sample volume
may therefore be less than 17 mL.
Density Gradient Medium
Density gradient medium refers to Lymphoprep™ (Catalog
#07801), Ficoll-Paque™ PLUS or other similar density gradient
media.
Recommended Medium
The recommended medium is phosphate-buffered saline with 2%
fetal bovine serum (PBS + 2% FBS, Catalog #07905).
Conversion of gto RPM
To convert gto rpm, use the following formula:
Where: RPM = centrifuge speed in revolutions per minute
RCF = relative centrifugal force (g)
Radius = radius of centrifuge rotor in centimeters (cm)
Troubleshooting
If the density gradient medium above the SepMate™ insert appears
red after centrifugation (i.e. some RBCs have not pelleted), the
SepMate™ tube can be spun at 1200 x gfor another 10 minutes
with the brake on. This situation may occur with samples that are
older than 24 hours.
Platelet Removal (optional)
Platelets present in the plasma layer may be removed from the
enriched MNCs in one of the following ways:
In step 5, pipette off the supernatant above the MNC layer
before pouring
In step 6, perform one of the washes at 120 x gfor 10 minutes
at room temperature, with the brake off
SUPPLEMENTARY PROCEDURE
Use of SepMate™ with RosetteSep™ Cocktails
SepMate™ tubes can be used with RosetteSep™ cell enrichment
cocktails to isolate specific cell types from human whole blood. For
available RosetteSep™ cocktails please refer to
www.rosettesep.com.
To use SepMate™ with RosetteSep™ cocktails:
1. Add RosetteSep™ cocktail to the whole blood sample using
volumes recommended in the RosetteSep™ cocktail Product
Information Sheet.
2. Incubate for 10 minutes at room temperature (15 - 25°C).
Note: The 10-minute incubation time is specific to this
procedure. It will have minimal effect on performance.
3. Follow the steps under SepMate™ Directions for Use, on
reverse page.
Note: Use density gradient medium recommended in the
RosetteSep™ cocktail Product Information Sheet.