Intracellular
Staining Techniques: Analysis of In Vivo Cytokine Production |
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View this document as a Microsoft Word file Flow cytometry is a powerful technique used to analyze cellular processes.
With antibodies specific for intracellular proteins, one can discern which
cells are activated by stimuli, and what cells synthesize in response
to stimulation. Fixed/permeabilized activated monocytes were used to study
activation by ?-interferon using antibodies to phospho-STAT1 [1]. Expression
of surface proteins accompanying activation can be identified and analyzed
by surface staining along with intracellular staining, allowing for identification
of cells from an organ. Moreover, cells from patients or animals can be
removed and analyzed during an on-going disease, allowing unique findings
in living organisms. None of these analyses are possible with cytokine-specific
ELISAs or bioassays. Examples of intracellular staining used in patient
cells are given in references [2,3]. For this chapter, we will concentrate
on the use of anti-cytokine antibodies in intracellular staining and analysis
by flow cytometry, and give examples of new information revealed by this
technique. Other methods for fixing and permeabilizing cells use methanol or formalin in buffer as the fixative [1], while Caltag sells a proprietary reagent which permanently permeabilizes cells once they have been treated. In our hands, the paraformaldehyde/DPBS was the best method for fixing, while the saponin buffer was the best method for permeabilization. For some intracellular antigens, such as phosphorylated signalling intermediates, the addition of methanol to the fixing procedure was found to enhance detection of signal over noise [1]. Thorough washing of cells is essential for low backgrounds; we always
did 3 washes, with the first wash including incubation in buffer for the
same length of time the cells were exposed to antibody or fluorochrome.
Furthermore, biotinylated antibodies for the primary staining gave lower
backgrounds than either unconjugated antibodies or F(ab)2' fragments. Generally, 0.1 ?g of antibody in 100 ?l Perm/Wash buffer/0.1% bovine
calf serum was sufficient to stain the cells, but each laboratory should
determine the optimal concentration for the antibodies used. Cells were
incubated with primary antibody for 45 min on ice. For the first wash
step, cells were incubated for 45 min in 1 ml of Perm/Wash buffer/0.1%
bovine calf serum before centrifugation (500g x 5 min); subsequent washes
were done without the long incubation time. After the final wash, cells
were resuspended in 0.1ml Perm/Wash buffer/0.1% bovine calf serum.
![]() Figure 1: Constitutive expression of OSM by bone marrow cells of B6 mice is revealed by intracellular staining of fixed/permeabilized cells, followed by analysis on a FACScan. Our laboratory studied the pathophysiology of the IL-6 cytokine family for some time; we used intracellular staining to study the pattern of induction of IL-6-like cytokine expression in hematopoietic organs of mice following tumor transplant. The induction was studied in SCID mice following intravenous administration of human myeloma, and also in C57BL/6 (B6) mice following subcutaneous injection of LL/2 squamous cell carcinoma. At times after administration of tumor, mice were euthanized; bone marrow and spleens were made into single cell suspension, and fixed/permabilized/stained as described to analyze the IL-6-type cytokine expression pattern. Biotinylated antibodies were purchased from R&D Systems, while streptavidin conjugates, fixative, and permeabilization reagents were from Pharmingen. In all figures, histograms show mean channel of fluorescence of antibody-stained sample in purple compared to streptavidin-PE-only-stained sample in green. Figure 1 shows that bone marrow of naïve C57BL/6 mice expressed high levels of OSM.
![]() Figure 2: IL-6 expression in spleen cells of SCID mice was induced by human myeloma cells, as revealed by intracellular staining for IL-6 on fixed/permeabilized cells. Analysis was performed on a FACScan, using CellQuest software for acquisition and analysis
Presence of tumors induced synthesis of IL-6 and OSM in tumor-free spleens of host mice; bone marrow cells constitutively expressed these cytokines, which are myeloma growth factors. It is possible that myeloma cells accumulate in bone marrow because growth factors present there induce proliferation [5]. These studies depended on intracellular staining to allow the analysis of proteins synthesized in tumor-bearing mice. This technique was used successfully in patients to study the types of T cells activated by rheuamtoid arthritis, allergic asthma, and atopic dermatits [7]. For a complete discussion on the detection of intracellular proteins by many methods, the reader is referred to the work of Bauer and Jacobberger [8]. There is much more information in this article than space allows here.
![]() Figure 3: Subcutaneous administration of LL/2 tumor cells in the flanks of B6 mice induced IL-6 expression in the spleen by day 7. Fixed/permeabilized spleen cells were analyzed on a FACScan after staining with biotinylated anti-mouse IL-6, followed by incubation with streptavidin-phycoerythrin, and analysis on a FACScan.
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