FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
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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
x
g
for 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
