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Document # CD-OP004 08/22© 2020 Lonza Walkersville, Inc.

PBMC Based T Cell Proliferation Assay
Instructions for use
Safety statements

THESE PRODUCTS ARE FOR RESEARCH USE ONLY. Not
approved for human or veterinary use, for application to humans
or animals, or for use in clinical or in vitro procedures.
WARNING: LONZA PRIMARY CELLS CONTAIN HUMAN
SOURCE MATERIAL; TREAT AS POTENTIALLY INFECTIOUS.
Each donor is tested and found non-reactive by an FDA-approved
method for the presence of HIV-I, hepatitis B virus and hepatitis Cvirus. Where donor testing is not possible, cell products are tested
for the presence of viral nucleic acid from HIV-1, hepatitis B virus,
and hepatitis C virus. Testing cannot offer complete assurance
that HIV-1, hepatitis B virus, and hepatitis C virus are absent. All
human-sourced products should be handled at the biologicalsafety level 2 to minimize exposure to potentially infectious
products, as recommended in the CDC-NIH manual, Biosafety in
Microbiological and Biomedical Laboratories, 5 th edition. If you
require further information, please contact your site safety officer
or Scientific Support.

Preparation of reagents for T cell assay

All work should be done in a laminar flow hood.
Decontaminate the external surfaces of all
supplement vials and the medium bottles withethanol or isopropanol.
1. IL-2 Medium
Make IL-2 media by adding 1 µL of 5 µg/mL IL-2
working solution to 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium for a finalconcentration of 5 ng/mL IL-2.
2. CFSE reagent
Follow company protocols for stock and working
solution of CFSE stain. Working solution should
be at final concentration of 5 µM.

PBMC Based T Cell Proliferation Assay

NOTE: All work is to be performed in a laminar flow hood. After
addition of CFSE, work should be protected from light.

1. Pipette 10 mL of pre-warmed X-VIVO® 15
Serum-free Hematopoietic Cell Medium into 15
mL conical tube and place in laminar flow hood.
2. Thaw PBMCs at 37oC for ~2 min until only asliver of ice remains and pipette cells into
X-VIVO® 15 Serum-free Hematopoietic Cell
Medium in 15 mL conical from step 1.

3. Wash cell vial with 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium from conicaland add back to conical.
4. Centrifuge cells at 300xg for 10 minutes at 4oC
5. Resuspend pellet in 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium and count using
Trypan Blue and a hemocytometer.
6. After counting, remove the needed number ofcells and place them into a new 50 mL conical.
For example, one reaction requires 1x106cells/mL. Adjust according to number ofreactions and reaction volume.
7. Add 1 mL CFSE 5 µM working solution per
1x106 cells and mix by vortexing
8. Incubate for 20 minutes protected from light atroom temperature.
9. After incubation, add X-VIVO® 15 Serum-freeHematopoietic Cell Medium at 4X the volume as
CFSE stain to stop the reaction.
10. Centrifuge cells 300xg for 10 minutes at room
temperature.
11. Resuspend cells in X-VIVO® 15 Serum-freeHematopoietic Cell Medium at a concentration of
10x106 cells/mL.
12. Incubate cells at room temperature for 10minutes.
13. During incubation step, prepare tissue culture
plates. For a 24-well plate, the following volumes
are recommended:
a. Negative control wells: 900 µL
X-VIVO® 15 Serum-freeHematopoietic Cell Medium
b. IL-2 control wells: 900 µL IL-2
Mediumc. Positive control wells: 850 µL IL-2
Medium + 50 µL CD3/CD28 Dyna
Beads (ThermoFisher 11131D)
14. After the 10 minute incubation from step 12, add
cells to each well (final concentration of 1x106
cells/mL – adjust according to tissue plate size).
For 24 well plates, add 100 µL cells.
15. Allow assay to incubate for 4 days at 37oC, 5%
CO2 to allow for T cell proliferation.

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16. On day 4, collect T cell suspension from culturesand stain with Live/Dead fixable cell dye for 20
minutes and analyze via FACS. A recommended
minimum of 100,000 cells for FACS analysis
should be taken into consideration.
17. Assess cells for CFSE intensity,decreased CFSE levels indicatesincreased T cell proliferation. .

Ordering information
Catalog no.

Description

SizeCC-2702

Human Peripheral ≥50 million cellsBlood
Mononuclear
Cells (hPBMC),
Cryopreserved

04-418Q

X-VIVO® 15
Serum -freeHematopoietic
Cell Medium

1L, complete with
L-Glutamine, gentamicin,and phenol red, xenofree

THESE PRODUCTS ARE FOR RESEARCH USE ONLY.
Not approved for human or veterinary use, for application tohumans or animals, or for use in clinical or in vitro procedures.

IL-2 (R&D Systems 202-IL-500) mentioned is a product from R&DSystems.
Dynabeads (ThermoFisher 11131D) mentioned is a product ofThermoFisher Scientific.
All other trademarks herein are marks of Lonza Groupor its subsidiaries.

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