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Protocol

General Approach to Adoptive Transfer and Cell Labeling

for Immunoimaging
Melanie P. Matheu, Michael D. Cahalan, and Ian Parker

INTRODUCTION
Imaging the single-cell dynamics of the immune system within an intact environment requires the
ability to look deep inside tissues and organisms with spatial and temporal resolutions adequate to
track cell morphology, motility, and signaling processes, all while minimizing perturbation of the sys-
tem under study. Fluorescence techniques are highly suited for this purpose, permitting both labeling
of specific cells, organelles, or proteins and functional readout of physiological events, and two-pho-
ton microscopy allows these processes to be visualized within native tissue environments. Adoptive
transfer, as described here, is the generally preferred method for introducing labeled cells of interest
into a host animal for immunoimaging. Cells are derived from a donor animal with a genetic back-
ground identical to that of the host and can either be endogenously fluorescent (e.g., isolated from a
transgenic mouse expressing the fluorescent protein) or can be labeled before transfer. Typically, trans-
ferring 2-6 × 106 labeled cells of a given type results in an appropriate cell density for two-photon
imaging.

RELATED INFORMATION
A general introduction to immunoimaging, as well as a brief discussion of the hardware required and
methods for quantitative analysis of multidimensional image stacks, are presented in
Immunoimaging: Studying Immune System Dynamics Using Two-Photon Microscopy (Matheu et
al. 2011a). Specific protocols are also available for Induction of an Immune Response for Imaging
Antigen-Presenting Cell/T-Cell Interactions (Matheu et al. 2011b), In Situ Lymph Node Imaging
(Matheu et al. 2011c), and In Vivo Lymph Node Imaging (Matheu et al. 2011d).

MATERIALS
CAUTIONS AND RECIPES: Please see the end of this protocol for appropriate handling of materials marked
with <!>, and recipes for reagents marked with <R>.

Reagents
Antibodies
These are used to check for cell population purity by fluorescence-activated cell sorting (FACS).
Cell separation kit
<!>Dye label, appropriate for animal model
Fetal bovine serum (FBS)
Lymphocytes

Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press,

Cold Spring Harbor, NY, USA, 2010.
Cite as: Cold Spring Harb Protoc; 2011; doi:10.1101/pdb.prot5565 www.cshprotocols.org

© 2011 Cold Spring Harbor Laboratory Press 180

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Isolate cells from the tissue of interest (e.g., lymph nodes, bone marrow) using a commercially available kit
(e.g., Matheu and Cahalan 2007). Cell lines can also be used (Matheu et al. 2008a). In vitro cultures of cer-
tain lymphocytes can be useful for the study of antigen presentation by monocyte-derived dendritic cells that
require several days to mature (Wei et al. 2007; Matheu et al. 2008b). Check all cells for population purity by
FACS analysis before adoptive transfer. Antibodies used for positive selection can alter the behavior of the iso-
lated lymphocyte population after injection (Garrod et al. 2007).
Medium, Roswell Park Memorial Institute (RPMI) 1640 or CO2-independent, serum-free
<!>Mice

Equipment
Centrifuge
Dissection tools
FACS cell sorter
Incubator preset to 37°C
Micropipettor and tips
Needles, 18-20-gauge
Syringes, sterile

METHOD

Cell Labeling

1. Label cells according to the conditions recommended in Table 1:

Endogenously fluorescent cells can be counterstained.

i. For amine- or thiol-reactive labels, treat cells in prewarmed (37°C) serum-free CO2-inde-
pendent medium or RPMI 1640.
ii. If imaging several days after cell injection, increase the dye-label incubation time by
10-20 min.
This can increase cell loss during the labeling process.

2. Wash the cells in 10 times the original volume with cell culture medium supplemented with
10% FBS.
3. Before injection, confirm by FACS that the intensity of the dye label is at least 50-fold above back-
ground cell fluorescence.
When using multiple fluorescent markers, adjust the loading conditions so that all labels are similarly bright.

4. Pellet the cells by centrifugation.

Inject cells into a host animal as soon as possible.

Cell Injection
Adoptively transfer cells using the appropriate injection route (Table 2). Premixing and coinjection of two or more labeled
cell types is useful to minimize the number of injections required and to synchronize the introduction of cells.

5. Resuspend cells in the desired volume for injection (Table 2).

6. Inject. Allow appropriate time for homing or immune response (Table 2).

Tissue Preparation for Imaging

7. When cells have equilibrated, harvest tissue for explant imaging, or prepare animal for intravital
imaging, as described in In Situ Lymph Node Imaging (Matheu et al. 2011c) and In Vivo Lymph
Node Imaging (Matheu et al. 2011d), respectively.

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Table 1. Commonly used fluorophores and labeling techniques for two-photon imaging

Probe class Labeling Labeling Duration and Representative probes

mechanism conditions example of use

Exogenous

Thiol-reactive Irreversible thiol cou- 37°C 5-15 MRPMI or 5-7 d Labeling for CMACa
pling following CO2-independent two-photon imaging CMFDACMTMR
esterase cleavage of medium (serum-
dye-label ester free)30-45 min

Amine-reactive Irreversible amine 37°C 2-8 MRPMI or 5-7 d Labeling for CFDA-SE
coupling following CO2-independent two-photon imaging; (CFSE)SNARF-1
esterase cleavage of medium (serum- Imaging dividing cell
dye-label acetate free)30-40 min populations; In vivo
labeling of DCs

Loading by mem- Passive diffusion of Follow product 1-4 h Fura-2 Indo-1

brane- permeant ester small molecule dye recommendations
into live cells; esterase
cleavage renders label
membrane-imperme-
ant

Active uptake and Peptide-conjugated s.c. injection of con- 1-3 d Labeling of Antigen-conjugated
internalization probe taken up/inter- centrated peptide/ local antigen-present- probe of choice
nalized by local anti- probe conjugate (up ing cells or antigen-
gen-presenting cells to 100 g/imaging presenting/collecting
site) at the site of cells in a draining
imaging or near a LN;Antigen distribu-
draining LN tion tracking

Dye-conjugated Diffusion, nonspecific i.v. or s.c. injection Tracking blood flow Texas Red, FITC, or
dextran blood or lymphatic (70-100 kDa, 0.5 rhodamine conju-
flow mg/100 L); Tracking gates
lymphatic flow
(20 kDa, 0.25 mg/
30-50 L)

QDs Free or antigen-conju- i.v. (20-100 L) or s.c. Blood flow and perfu- QDs + antigen of
gated (20 pM in 50 L) sion monitoring; choice
injection Induction of an
immune response/
antigen distribution
tracking

Carbohydrate binding Sialic acid and lectin- Soak tissue in 100 Same-day labeling WGA (Alexa 633)
(whole-tissue label- binding dye-conju- g/mL lectin (pre- and imaging to
ing) gated proteins pared in RPMI or define tissue struc-
CO2-independent tures in vitro
medium) for 2-4 h at
4°C
(continued)

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Table 1. Continued
Probe conjugated to Labeling of specific Preinjection of 100- Labeling cells and tis- Antibody-probe
antibody cells in live tissue 200 g label-conju- sues; likely varies with conjugate
(presumably by anti- gated antibody near antibody and probe
body binding to cell the draining LN of set used (not tested
surface epitopes) interest extensively)

Endogenous

Promoter-driven FP Cell-specific label; All Produce transgenic Lifetime of the cell Various FPs (e.g., CFP,
expression cell types (when FP is animal or order read- (label is permanent, GFP, YFP)
expressed under a ily available animal nontoxic) Adoptive mCherryHcRedKaede
ubiquitously model (Some pro- transfer of purified
expressed protein moters might not cells; Imaging
promoter) induce high enough endogenous cell
FP expression to be behavior;Production
visible with two-pho- of chimeric animals
ton imaging) (Witt et al. 2005);
Photoconvertible FPs
for tracking the
behavior ofsingle cells
(Tomura et al. 2008)

SHG Endogenous highly Emission best when Long-term imaging of Myosin, collagen, and
ordered tissues emit excitation is perpen- endogenous struc- tendon
wavelength ~ half of dicular to structure tures in various tissues
two-photon excita- orientation. Excitation (e.g., LN, skin, lung,
tion at 880-920 nm is pre- muscle)
ferred forblue/violet
channel detection

a
Abbreviations: CFDA-SE, carboxyfluorescein diacetate succinimidyl ester; CFP, cyan fluorescent protein; CFSE, carboxyfluorescein
succinimidyl ester; CMAC, 7-amino-4-chloromethylcoumarin; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5-(and -6-)-
{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine; DC, dendritic cell; FITC, fluorescein isothiocyanate; FP, fluorescent pro-
tein; GFP, green fluorescent protein; i.v., intravenous; LN, lymph node; QDs, quantum dots; s.c., subcutaneous; SHG,
second-harmonic generation; SNARF-1, seminaphtharhodafluor-1; WGA, wheat germ agglutinin; YFP, yellow fluorescent protein.

ACKNOWLEDGMENTS
We thank Luette Forrest for helpful comments as well as past members of the Cahalan Laboratory,
especially Mark J. Miller and Sindy H. Wei, for their valuable contributions to this protocol.

REFERENCES
Garrod KR, Wei SH, Parker I, Cahalan MD. 2007. Natural killer cells actively Cold Spring Harb Protoc doi: 10.1101/pdb.prot5566.
patrol peripheral lymph nodes forming stable conjugates to eliminate Matheu MP, Cahalan MD, Parker I. 2011c. In situ lymph node imaging.
MHC-mismatched targets. Proc Natl Acad Sci 104: 12081–12086. Cold Spring Harb Protoc doi: 10.1101/pdb.prot5567.
Matheu MP, Cahalan MD. 2007. Isolation of CD4+ T cells from mouse Matheu MP, Cahalan MD, Parker I. 2011d. In vivo lymph node imag-
lymph nodes using Miltenyi MACS purification. J Vis Exp 2007: 409. ing. Cold Spring Harb Protoc doi: 10.1101/pdb.prot5568.
Matheu MP, Beeton C, Garcia A, Chi V, Rangaraju S, Safrina O, Tomura M, Yoshida N, Tanaka J, Karasawa S, Miwa Y, Miyawaki A,
Monaghan K, Uemura MI, Li D, Pal S, et al. 2008a. Imaging of effec- Kanagawa O. 2008. Monitoring cellular movement in vivo with
tor memory T cells during a delayed-type hypersensitivity reaction photoconvertible fluorescence protein “Kaede” transgenic mice.
and suppression by Kv1.3 channel block. Immunity 29: 602–614. Proc Natl Acad Sci 105: 10871–10876.
Matheu M, Sen D, Cahalan MD, Parker I. 2008b. Generation of bone Wei SH, Safrina O, Yu Y, Garrod KR, Cahalan MD, Parker I. 2007. Ca2+
marrow derived murine dendritic cells for use in 2-photon imaging. signals in CD4+ T cells during early contacts with antigen-bearing
J Vis Exp 2008: 773. dendritic cells in lymph node. J Immunol 179: 1586–1594.
Matheu MP, Cahalan MD, Parker I. 2011a. Immunoimaging: Studying Witt CM, Raychaudhuri S, Schaefer B, Chakraborty AK, Robey EA.
immune system dynamics using two-photon microscopy. Cold 2005. Directed migration of positively selected thymocytes visual-
Spring Harb Protoc doi: 10.1101/pdb.top99. ized in real time. PLoS Biol 3: e160. doi: 10.1371/
Matheu MP, Cahalan MD, Parker I. 2011b. Induction of an immune journal.pbio.0030160.
response for imaging antigen-presenting cell/T-cell interactions.

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CAUTIONS
[NOTE: For reagents marked with the <!> symbol not listed below, please consult the manufacturer’s Material Safety Data Sheet
for further information.]

Animal treatment
Procedures for the humane treatment of animals must be observed at all times. Consult your local animal facility
for guidelines.

Dyes
Follow manufacturer’s safety guidelines.

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General Approach to Adoptive Transfer and Cell Labeling for Immunoimaging

Melanie P. Matheu, Michael D. Cahalan and Ian Parker

Cold Spring Harb Protoc; doi: 10.1101/pdb.prot5565

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