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Document # CD-OP003 08/22© 2020 Lonza Walkersville, Inc.

Dendritic Cell (DC) and T Cell Assay from Matched PBMCs
Instructions for use
Safety statements

and GM-CSF (800 U/mL). Only make enoughculture base medium as needed. Oncecytokines are added to medium do not store
media for longer than 30 minutes. Media
supplemented with cytokines should be madefresh each time prior to use.
4. IL-2 MediumPrepare stock IL-2 media by adding 1 µL of 5µg/mL working solution IL-2 to 1 mL X-VIVO™ ®
15 for a final concentration of 5 ng/mL.
5. CFSE Reagent
Follow company protocols for stock and working
solutions of CFSE stain. Working solution should
be at a final concentration of 5 µM.

Preparation of reagents

Culturing and maturation of DCs

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.

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. Release buffer
Prepare a stock bottle of 5 mM EDTA in PBS
without Ca2+ or Mg2+ by aseptically adding 10
mL of 0.5 mM EDTA to 990 mL PBS. This buffer
can be stored at 2-8oC for 1 year.
2. Adherence Medium
Supplement X-VIVO® 15 Serum-free Hematopoietic
Cell Medium with 0.5% heat inactivated humanplasma containing 50 U/mL Heparin. Keep media at4°C.
3. DC Culture Medium Base
Prepare culture medium base during incubation
for plastic adherence. Do not add cytokines toculture medium base more than 30 minutes
prior to completion of plastic adherence
incubation. Supplement X-VIVO® 15 Serumfree Hematopoietic Cell Medium with 0.5% heat
inactivated human plasma containing 50 U/mLHeparin. Add the following cytokines to theculture medium base such that the final
concentration is as follows: IL-4 (1000 U/mL)

NOTE: All work is to be performed in a laminar flow hood.

1. Pipette 10 mL of pre-warmed X-VIVO® 15Serum-free Hematopoietic Cell Medium into a
15 mL conical tube.
2. Thaw PBMCs in a water bath at 37oC for ~2 min
and pipette cells into the X-VIVO® 15 Serumfree Hematopoietic Cell Medium in the 15 ml
conical from step 1.
3. Wash cell vial with 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium and add wash
to 15 mL conical from step 2.
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 count, plate PBMCs at 5x106 cells/mL inadherence medium into appropriate flask. Adjust
accordingly to flask size.
7. Place the planted flasks at 37oC ± 2oC, 5% CO2± 2% CO2 for 2 hours for adherence. Verify
adherence of cells under the microscope. Cells
should not be free floating or move with themedia as the plate is inspected under themicroscope.
8. Rinse the adherent cells with PBS (without Ca2+,Mg2+, or EDTA as it will cause cells to detach),page 1 of 3

by gently adding PBS to flask without disturbing
attached cells. Gently tap flask and swirl washaround the flask.
9. Repeat wash for a total of 2 washes per flask.
10. Feed adherent cells with culture medium basecontaining cytokines. For 24 well plates use 1
mL culture medium base containing cytokines.
Adjust volume of culture medium basecontaining cytokine accordingly. Place the flasks
at 37oC ± 2oC, 5% CO2 ± 2% CO2 for 3 days.
11. On day 3, collect media and nonadherent cellsand spin at 300xg for 10 minutes.
12. Resuspend cell pellet in 1 mL culture medium
base containing cytokines and reseed back intoculture.
13. To mature and activate the DCs on day 3, add
100 µg/mL LPS to each culture. Place the flasks
at 37oC ± 2oC, 5% CO2 ± 2% CO2 for 24 hours.

DC:T Cell 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 intoa 15 mL conical.
2. Thaw PBMCs at 37oC for ~2 min and pipettecells into X-VIVO® 15 Serum-freeHematopoietic Cell Medium in the 15 mL
conical from step 1.
3. Wash cell vial with 1 mL X-VIVO® 15
Serum-free Hematopoietic Cell Medium andadd wash to conical.
4. Centrifuge cells at 300xg for 10 minutes at4oC.
5. Resuspend pellet in 1 mL PBS and countusing Trypan Blue and a hemocytometer.
6. Purify CD4+ T cells using Miltenyi CD4
Microbeads and following manufacturer’sprotocol.
7. Once purified, resuspend cells in 1 mL PBS
and count using Trypan Blue and ahemocytometer.

Note: Pre-isolated human dendritic cells (Cat: CC2701) are available for purchase from Lonza. If
using Lonza dendritic cells, start protocol from step
13. Use of pre-isolated Lonza dendritic cells results
in a reduction of time of 3 days.

Note: Pre-isolated human naïve CD4 T cells (Cat:4W-202) are available for purchase from Lonza. Ifusing Lonza naïve CD4 T cells, thaw the cells asdescribed in steps 2-5 and proceed with protocol
from step 8. Use of Lonza naïve CD4 T cells results
in a reduction of reagents and time.

Figure 1: Mature DC morphology after 24 hour
exposure to LPS. Mature DCs should have a roughsurface with multiple pseudopodia.
14. Prior to harvest on day 4, ensure that PBS
without Ca2+ or Mg2+ plus 5 mM EDTA is icecold. Remove culture medium and any
unattached cells from each flask.
15. Briefly rinse flasks with ice cold PBS withoutCa2+ or Mg2+ plus 5 mM EDTA. Place rinse inconical with the media collected from step 14.
16. Add additional ice-cold PBS without Ca2+ orMg2+ plus 5 mM EDTA to each flask andincubate for 10 minutes.
17. After incubation, tap sides of flask and washflask to assist with detachment. Remove all
detached cells and place them in a conical tube.
Centrifuge cells at 300xg for 10 minutes.
18. Resuspend cells in 1 mL X-VIVO® 15 Serumfree Hematopoietic Cell Medium.

8. After count, remove appropriate number of cells
and place into a new 50 mL conical for CFSEstaining. For example, one reaction requires
1x106 cells/mL. Adjust according to number ofreactions and reaction volume.
9. Add 1 mL CFSE working solution (5 µM) per
1x106 cells and mix by vortexing.
10. Incubate 20 minutes protected from light at RT.
11. After incubation, add X-VIVO® 15 Serum-freeHematopoietic Cell Medium at 4X the volume as
CFSE stain to stop the reaction. Incubate for 5
minutes at room temperature.
12. Centrifuge cells 300xg for 10 minutes. Once Tcells have been stained, allow the cells to rest atroom temperature a minimum of 10 minutes
prior to stimulation.
13. During incubation, prepare assay plate. For a24-well plate the following volumes and controls
are recommended:
a. Negative control wells: 900 µL XVIVO® 15 Serum-freeHematopoietic Cell Medium
b. Positive control wells: 850 µL IL-2
Medium + 50 µL CD3/CD28 Dyna
Beads (ThermoFisher 11131D)
c. DC and T cell wells: Co-culture
stained T cells with mature DCs at apage 2 of 3

ratio 10:1 (T cell: DC) in 800 µL
X-VIVO® 15 Serum-freeHematopoietic Cell Medium
d. DC and T cell and IL-2 wells: Coculture stained T cells with mature
DCs at a ratio 10:1 (T cell: DC) in800 µL IL-2 Medium
14. Add T cells to each well (final concentration of
1x106 cells/mL – adjust according to tissue platesize). For 24 well plates, add 100 µL cells.
15. Add DCs to each well (final concentration of
1x105 cells/mL – adjust according to tissue platesize). For 24 well plates, add 100 µL cells.
16. Allow assay to incubate for 4 days at 37oC, 5%
CO2 to allow for T cell proliferation.
17. On day 4, collect T cell suspension from culturesand stain with Live/dead fixable cell dye for
20 minutes and analyze via FACS.
18. Assess cells via CFSE intensity (MFI of FITCchannel) to determine T cell proliferation (i.e.,decreased CFSE signal indicates high T cellproliferation)

LPS (Sigma L5668-2ML) mentioned is a product from Millipore
Sigma.
GM-CSF (PeproTech 300-03) mentioned is a product from
PeproTech.
IL-4 (R&D Systems 204-IL-010) mentioned is a product from R&DSystems.
IL-2 (R&D Systems 202-IL-500) mentioned is a product from R&D
Systems.
Dyna Beads (ThermoFisher 11131D) mentioned is a product ofThermoFisher Scientific.
All other trademarks herein are marks of Lonza Groupor its subsidiaries.

Note: Assay can be performed with pre-isolated Lonza dendriticcells (Cat: CC-2701) and Lonza CD4 + naïve T cells (Cat: 4W202). Ideally allogeneic or autologous matched cells would beneeded to perform the assay in this manner.

Ordering information
Catalog no.

Description

SizeCC-2702

Human Peripheral ≥50 million cells
BloodMononuclearCells (hPBMC),Cryopreserved

CC-2701

NHDC- Human
Dendritic Cells≥2.5 million cells

4W-202

Human
CD4+/CD45RA+Naïve T cells,
cryopreserved≥5 million cells

04-418Q

X-VIVO™ ® 15
Serum -freeHematopoietic
Cell Medium

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

BE17-516Q

Phosphate
Buffered Saline(1X) without
Calcium andMagnesium

1L

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.
CD4 MicroBeads, human (Miltenyi Biotec 130-045-101)
mentioned is a product from Miltenyi Biotec.

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