| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Abstracts of Oak Ridge Posters |
1 Molecular Staging, Inc., Cellular Analysis Section, Flow Cytometry Group, 300 George St., Suite 701, New Haven, CT 06511
aauthor for correspondence: fax 203-776-5278, e-mail ragoor{at}molecularstaging.com
Rolling circle amplification (RCA) generates a localized signal via isothermal amplification of an oligonucleotide circle reporter sequence. The application of this approach to flow cytometry could extend the utility of existing methods by enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer and of hematologic and other abnormalities.
RCA technology is applicable to a variety of platforms for the simultaneous detection of molecules as a function of either antigenicity or nucleic acid sequence (1)(2)(3)(4). In flow cytometry, cells of interest are characterized based on population gating. Efficient gating strategies are crucial for accurate immunophenotyping, more so in a heterogeneous cell suspension such as peripheral blood mononuclear cells (PBMCs). Usually a combination of light scatter (forward and side) and immunophenotypic markers is critical in identifying the specific cells of interest. A panel of antibodies is usually used to characterize a subset of cells based on their surface markers. However, cells can be best characterized only when the staining for each of these markers is bright enough to clearly differentiate them from unstained cells. This requires specific antibodies and intense detection signals. Abundantly expressed cell surface markers are not difficult to stain and identify compared with rare surface antigens, which are currently gaining importance in diagnostics and clinical studies. Therefore, the common challenge in clinical or diagnostic flow analysis is insufficient signal (low-intensity signals) leading to inefficient use of the existing antibody library.
RCA technology can help overcome these problems. The RCA technology (RCATTM) also allows for multiplexing or multiparametric analysis of various markers simultaneously, supporting the expanding use of complex marker panels for disease diagnosis and prognosis. RCA-mediated signal amplification has been successfully applied to the detection of cell surface antigens (e.g., CD4 and CD28) on PBMCs. This technical report describes the technology and protocol for flow cytometric analysis of lymphocyte surface markers. We have achieved a >10-fold increase in median fluorescent intensity (MFI) with RCA compared with conventional detection methods.
Human PBMCs were separated from whole blood by density gradient centrifugation using Vacutainer CPT tubes (Becton Dickinson). Two low-speed (300–400g) washes were performed in 1x phosphate-buffered saline (PBS; pH 7.4) to minimize platelet contamination. After centrifugation, cells were diluted to obtain 
5 x 106 cells so that 100 µL would give 500 000 cells/sample.
Although this may be a limitation in studies requiring recovery of viable cells, we found that cell fixation and permeabilization play an important role in cell staining and RCA. Although fixation was not absolutely necessary for amplification of signals, a light fixation substantially enhanced the efficiency of the subsequent RCA reaction. Therefore, for the studies described here, 500 000 cells were fixed with 5 g/L paraformaldehyde for 2 min at room temperature. The cells were then washed and blocked with 50 mL/L normal goat serum (NGS) in PBS, pH 7.4. An equal volume of 50 mL/L NGS, isotype controls (negative control samples), or 2 mg/L primary antibody (phycoerythrin-conjugated anti-CD4/CD28) diluted in 50 mL/L NGS was added to the cells and incubated in the dark for 30 min at room temperature. Alternatively, samples to be detected by second-step reagents were incubated with a biotinylated anti-CD4/CD28 antibody (30 min at room temperature), washed in PBS, and resuspended in a 1:1000 dilution of phycoerythrin-conjugated streptavidin (cat. no. S-866; Molecular Probes) for 15 min at room temperature (in the dark). After this incubation, cells were washed, centrifuged, and kept on ice until analysis by flow cytometry.
RCA.
The blocked PBMCs were incubated with a preannealed complex of an anti-CD4–primer 4.2 conjugate [details of conjugates and circle are described in Gusev et al. (4)] and circle 4.2 in the conjugate-circle-complex diluent [C3-diluent; containing 150 mmol/L potassium glutamate, 10 mmol/L HEPES (pH 7.4), and 5 mL/L polyvinyl alcohol] for 30 min at room temperature. The negative controls were incubated with circle only (without conjugate) in the C3-diluent. Preannealing was done with 100 mg/L antibody conjugate and 2000 nmol/L DNA circle in the C3-diluent for 15 min at 37 °C, and the mixture was diluted 50-fold (in C3-diluent) before addition to cells. Alternatively, for indirect detection followed by RCAT analysis, the preannealed complex was prepared with an anti-biotin–primer 4.2 conjugate and circle 4.2, diluted 50-fold in C3-diluent, and added for 5 min at room temperature directly to PBS-washed cells that were incubated with a biotinylated primary antibody.
After the preannealed conjugate/circle incubations, the cells were washed and subjected to RCAT in the presence of 30 U of 
29 polymerase per 25-µL reaction volume. The reaction mixture contained 150 mmol/L potassium glutamate, 35 mmol/L HEPES (pH 7.4), 10 mmol/L magnesium acetate, 7 mmol/L dithiothreitol, 70 mg/L bovine serum albumin, and 400 µmol/L each deoxynucleotide triphosphate. The reaction was incubated at 31 °C for 15 min. The cells were then centrifuged with an addition of 100 µL of 50 mL/L NGS in PBS, pH 7.4, and decorated in 50 µL of an 8 mg/L phycoerythrin–decorator complex in 50 mL/L NGS.
The decorator used was an oligonucleotide complementary to the DNA circle 4.2 and had a biotinylated thymidine at the 3' end with an eight-nucleotide spacer of deoxyinosine and deoxyuridine. This decorator was incubated with phycoerythrin-conjugated streptavidin overnight at 4 °C in PBS, pH 7.4, and purified on a size-exclusion column to remove the free constituents, essentially as done by Davis et al. (5) with minor modifications.
Flow cytometry.
Cells were analyzed by use of a Becton Dickinson FACSCalibur equipped with a 15 mW, 488 nm, air-cooled argon ion laser; a four-color analytical module; and a 635 nm red diode laser for Cy5 studies. Ten thousand events were acquired in list mode while gating lymphocytes and excluding debris. A histogram overlay of antibody fluorescence was constructed on the gated regions, and values for median channel fluorescence of CD4 expression were derived from the linear scale (1–10 000). The increase in amplification was calculated by the fold difference in the signal-to-noise ratios of conventional and RCAT detection. Data were analyzed using CELLQuest 3.3 (Becton Dickinson), WinMDI (freeware), and MS Excel.
During the development of the described protocol, a series of experiments were performed. Conditions for fixation of cells, application of RCA without destroying the antigenicity of cells, and the amplification step as such were adjusted to optimize the performance of the technology. We compared the proposed method with existing conventional detection methods to demonstrate the performance of the technology; experiments were repeated multiple times (n = 54 for CD4 and n = 12 for CD28 experiments) to determine reproducibility. Reproducibility was determined by the calculating the fold amplification (signal-to-noise ratio). The CV was <0.02%.
With conventional detection methods, the lymphocyte profile for CD4 is seen as a tight peak at the third log of signal intensity. When comparing MFIs with RCA, however, we saw a >10-fold increase in signal with minimal increase in the background peak in both the direct and indirect detection systems (Table 1
and Fig. 1
).
|
|
CD4 is highly expressed (98 000 molecules/cell), but only 12 000 molecules of CD28 antigen are expressed in lymphocytes (6). Therefore, we chose CD28 as a second marker with moderate expression on a T-cell subset. Results showed a >10-fold increase in fluorescence intensity by signal amplification using RCA compared with conventional indirect detection using streptavidin-phycoerythrin (Table 1
). Signal amplification was also effective with CD4 detection on monocytes (moderate expression, usually approximately one log lower than on lymphocytes), going beyond a 10-fold increase over direct labeling (data not shown).
Our results demonstrate the potential of RCAT in enabling the development of a next-generation tool for better detection and monitoring of cell surface markers in pathologic conditions by flow cytometry. This can be achieved because of the increased sensitivity that the technology offers to the process of signal amplification. Unlike PCR or tyramide signal amplification, RCA requires only a single round of amplification and the newly synthesized product remains attached to the target, providing localized specific signals. In addition, the technology can be rapidly adapted to other clinical testing modalities, such as beads, tissues, plates, and microarrays. RCAT offers potential in developing accurate, quantitative, and standardized evaluation of individual immunologic responses, which has become increasingly important with the advancement of several immunotherapeutic interventions to clinical trials in recent years.
Acknowledgments
We acknowledge Dave Riches, Mehul Patel, Edward Zelazny, Dr. Osama Alsmadi, and Dr. Vanessa Wheeler for their contributions.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
