Positive Selection
Catalog #18782
EasySep™ Mouse CD25
Regulatory T Cell Positive
Selection Kit
Instructions for use with (Catalog #):
 EasyEights™ Magnet (18103)
FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
Description
This product information sheet (PIS) outlines directions for use with the following EasySep™ magnets:
EasyEights™ EasySep™ Magnet (Catalog #18103)
If using other EasySep™ magnets, or for further information on sample preparation, components, stability, storage, and purity assessment, please refer to the PIS
(Document #28070) included with the kit, or download a copy from our website www.stemcell.com, or contact Technical Support at [email protected].
Notes and Tips
Recommended Medium
EasySep™ Buffer (Catalog #20144), RoboSep™ Buffer (Catalog #20104), or phosphate-buffered saline (PBS) containing 2% fetal bovine serum
(FBS; Catalog #07905) and 1 mM EDTA. Medium should be free of Ca2+ and Mg2+.
Sample Preparation
Prepare the sample as a single-cell suspension at the listed concentration in recommended medium (see Table 1 and Notes and Tips). Cells must be placed in
the required plasticware to properly fit into the appropriate EasySep™ magnet.
Note: For detailed instructions on how to prepare the sample, refer to the PIS (Document #28070) included with the kit.
Table 1. Sample Preparation
EASYSEP™ MAGNETS
EasyEights™
PROTOCOL
Required
Plasticware
Start Cell
Concentration
Start Sample Volume
(Cell Number)
5 mL tube
14 mL tube
5 mL (12 x 75 mm) polystyrene round-bottom tube
(e.g. Corning® Catalog #352058)
14 mL (17 x 100 mm) polystyrene round-bottom tube
(e.g. Corning® Catalog #352057)
1 x 108 cells/mL
1 x 108 cells/mL
0.5 - 1.5 mL per tube
(0.5 - 1.5 x 108 cells per tube)
1 - 8 mL per tube
(1 - 8 x 108 cells per tube)
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Document #28070SM | Version 1_0_0
EasySep™ Mouse CD25 Regulatory T Cell Positive Selection Kit
Positive Selection
Directions for Use
Prepare sample as directed in Table 1 (see Sample Preparation). Refer to Table 2 for detailed instructions regarding the EasySep™ procedure compatible with each magnet.
Table 2. EasySep™ Mouse CD25 Regulatory T Cell Positive Selection Kit Instructions
EASYSEP™ MAGNETS
EasyEights™
STEP
INSTRUCTIONS
5 mL tube
14 mL tube
50 μL/mL of sample
50 μL/mL of sample
RT for 5 minutes
RT for 5 minutes
50 μL/mL of sample
50 μL/mL of sample
RT for 10 minutes
RT for 10 minutes
20 μL/mL of sample
20 μL/mL of sample
RT for 5 minutes
RT for 5 minutes
30 seconds
30 seconds
20 μL/mL of sample
20 μL/mL of sample
Mix and incubate.
RT for 5 minutes
RT for 5 minutes
Add recommended medium to top up the sample to
the indicated volume. Mix by gently pipetting up
and down 2 3 times.
Top up to 2.5 mL
Top up to 5 mL for start sample < 4 mL
Top up to 10 mL for start sample  4 mL
Place the plasticware (without lid) into the magnet
and incubate.
RT for 15 minutes
RT for 15 minutes
Discard supernatant
Discard supernatant
Steps 6 and 7, two more times
(total of 3 x 15-minute separations)
Steps 6 and 7, two more times
(total of 3 x 15-minute separations)
Isolated cells are ready for use
Isolated cells are ready for use
Add FcR Blocker to sample.
1
Mix and incubate.
Add Positive Selection Cocktail to sample.
2
Mix and incubate.
Add PE Selection Cocktail to sample.
3
Mix and incubate.
4
Vortex RapidSpheres™.
Add RapidSpheres™ to sample.
5
6
7
Carefully pipette** (do not pour) the supernatant
and discard.
8
Repeat as indicated.
9
Remove the plasticware from the magnet and
resuspend cells in appropriate amount of desired
medium.
RT; room temperature (15 - 25°C)
**It is recommended to collect the unwanted cell-suspension, all at once, into a single pipette (e.g. for the EasyEights™ 5 mL tube use a 2 mL serological pipette and for the EasyEights™ 14 mL tube use a 10 mL serological pipette)
Copyright © 2014 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasyEights, EasySep, and RapidSpheres are trademarks of STEMCELL Technologies
Inc. Falcon is a trademark of Corning® Incorporated. All other trademarks are the property of their respective holders.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485 MEDICAL DEVICE STANDARDS.
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Document #28070SM | Version 1_0_0