2008 PBL Cat_mech_r.qxd:PBL Catalog.mech 11/29/07 1:51 PM Page 48
Technical Appendix
Flow Cytometry
PBL InterferonSource offers high-quality fluorochrome-conjugated fluorescein 6) Gently mix to resuspend cells in 40 µl of wash buffer
isothiocyanate, (FITC) and R-phycoerythrin (R-PE) antibodies designed to 7) Block nonspecific binding sites (FcR) by adding 10 µl rabbit IgG (Sigma
monitor the presence of cytokines and cytokine receptors. Aldrich, I-8140).
Flow cytometry uses the principles of light scattering, light excitation, and 8) Incubate 5 min at room temperature.
emission of fluorochrome molecules to determine characteristics of an 9) Add the MAbs as follows:
individual particles or cells by examining how they flow in liquid. Fluorescence- a) Tubes 1 and 2: 3 µl negative control mouse IgG
2a
(DakoCytomation
activated cell-sorting (FACS) is a specialized type of flow cytometry. The (XO943), final concentration 6 µg/ml)
instruments collect qualitative and quantitative information by measuring b) Tubes 3 and 4: Anti-IFNAR2 MAb (PBL product #21385-1) to a final
visible and fluorescent light emissions. Flow cytometry is a procedure to concentration of 2.5 µg/ml.
rapidly obtain detailed information of complex populations of cells or cellular 10) Incubate on ice for 40 min.
constituents. 11) Wash twice in wash buffer.
Immunologists often use FACS to sort cells using conjugated fluorochromes 12) Resuspend cells in 50 µl wash buffer with biotin-conjugated goat
to antibodies or fluorochromes that can bind to other molecular targets, such as anti-mouse IgG (Jackson ImmunoResearch 115-066-072) to a final
DNA, cytokines, or other proteins. Typically, the data is analyzed and presented concentration of 5 µg/ml.
in the form of a one or two-dimensional dot plots or as a single-dimension 13) Incubate on ice for 40 min.
histogram. 14) Wash twice in wash buffer.
15) Resuspend cells in 50µl wash buffer to include
Principle a) Tubes 1-4: R-Phycoerythrin-conjugated Streptavidin (Jackson
Flow cytometers use hydrodynamic focusing to align and separate populations ImmunoResearch 016-110-084) to a final concentration of 2.5 µg/ml.
of cells into a stream that travels as a single-file of particles. The sample is Further, add to
injected into the center of a sheath of a slow moving liquid which is surrounded i. Tubes 1 and 3: 2 µl negative control mouse IgG
1
-APC
by an outer sheath of faster flowing fluid. This allows cells in suspension to be (DakoCytomation (XO968), final concentration 4 µg/ml) .
drawn into a laminar stream allowing the cells to pass individually through the ii. Tubes 2 and 4: 1 µg/ml final concentration APC-conjugated
detection region. mouse anti-human CD3 MAb (Ancell, 144-060).
At the measuring region, a beam of monochromatic light, usually from a 16) Incubate on ice for 15 min.
laser, intersects the cells. Emitted light is deflected in all directions by the cell 17) Wash cells twice in wash buffer.
and is collected using optics that aligns the light to pass through a series of 18) Resuspend cells in PBS plus 1 µg/ml propidium-iodine (to exclude dead
filters and dichroic mirrors that separate specific wavelengths. Forward scatter cells from the measurements. This step can be omitted).
(measured on the same axis as the laser beam) correlates with cell surface 19) Harvest cells for flow cytometry: collect at least 50000 events contained
area and size and is a measurement of diffraction. Side scattered light in the live cells gate.
(measured at 90 degrees to the laser beam) is a result of refraction and is
correlates to internal cellular granularity.
To detect specific cellular molecules, fluorescent probes containing
covalently attached fluorochromes, such as fluorescein or R-phycoerythrin are
used. The emission spectra of these fluorochromes is different which allows for
the identification of cells based upon the color of the emitted light, The emitted
light is detected, amplified, then converted into an digital signal which is
subsequently analyzed by computer.
End-User Protocols
(Courtesy of Dr. Laura Conti, Center for Experimental Research and Medical F
L4-H
Studies, San Giovanni Battista Hospital, Turin, Italy). Protocol not tested in PBL’s laboratories.
Flow Cytometric Analysis of IFNAR2 Surface Expression on Human Peripheral
Blood Mononuclear Cells (PBMC)
1) Isolate peripheral blood mononuclear cells (PBMC) from heparinized FL2-H
venous blood obtained from healthy donors by Ficoll gradient (Type 400,
Pharmacia, Uppsala, Sweden) centrifugation.
FL2-H = IFNAR2
2) Collect
FL4-H = CD3
a) Anti-IFNAR2 MAb (PBL product #21385-1)
b) Isotype-matched negative control MAb (e.g., mouse IgG
2a
, Flow cytometric analysis of PBMC isolated from venous blood of healthy
DakoCytomation XO943). donors and promptly stained for IFNAR2 (FL2-H) and CD3 (FL4-H) as
3) Wash cells twice in NaCl. (Resuspend gently, then centrifuge at 4°C, 1800 described. The quadrant markers were set on the bases of the fluorescence
rpm 5 min). of the same PBMC stained with the corresponding isotype-matched
4) Collect 10
6
cells per FACS tube and wash with wash buffer. negative control antibodies. Numbers represent the quadrant percentiles of
5) Carefully pipette off the supernatant and discard. cells staining positively for the indicated molecules.
PBLInterferonSource Toll Free: 1 877- PBL-8881
48
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