Sony Biotechnology Inc. Application Data, SA38 Spectral Analyzer TBNK Panel Page 1 TBNK panel on the SA38 Spectral Analyzer TBNK (B-Cell, T-Cell, and natural killer cells) panels are frequently used to examine major leukocyte populations. All of these populations are important for normal immune function. With the ability to analyze more fluorochromes, the SA38 allows additional subsets to be defined making the most of your precious samples. In this example two additional markers were added to the traditional panel, CD14 ( monocyte marker) and CD7 ( lymphocyte marker). The NK cell population is traditionally defined by a heterogeneous expression of the CD16 and CD56 population. The addition of CD7 helps identify the T cell subset of the activated NK population.1 In this experiment a TBNK panel was run on a Sony SA38 spectral analyzer. The spectral analyzer has several advantages over a traditional flow cytometer. PMT layout and spectral unmixing allow the detection of overlapping fluorochromes Enhancement of dim signal detection for better visualization of populations Complete data views that enable the viewing of the entire panel in a single graph The SA38 software creates traditional bivariant dot plots in addition to spectral plots. In this example traditional analysis will be presented first and then combined with spectral analysis to emphasize the clarity that spectral plots offer to complex data analysis. Specificity Fluorochrome Purpose/Identifies Cat. No. CD3 All T-cells 218653 CD4 PE-AlexaFluor 7 CD4 helper T-cell N/A CD8 Brilliant Violet 57 CD8 effector T-cell 21519 CD19 Brilliant Violet 65 B-cells 21112 CD16 Brilliant Violet 421 NK-cells 21119 CD7 NK cell subset 231554 CD14 -Cy 7 Monocytes 2191 CD45 Alexa Fluor 532 Gating/Leukocytes N/A A B 1 8 6 Traditional Flow Cytometer 44 46 48 5 5 54 56 58 6 6 64 66 68 7 7 74 76 78 8 SA38 Spectral Analyzer TBNK Panel 4 CD16/56 CD3 CD45 Alexa 532 CD8 BV57 CD19 BV65 CD7 CD4 PE-Alexa 7 CD14 V1 V2 1 2 3 4 5 6 7 8 9 1 12 13 14 15 16 17 18 19 21 22 23 24 25 26 27 28 29 3 31 32 PMT Channel Number Spectral profiles of fluorochromes used in the TBNK panel (above) by wavelength (A) and channel number (B). The SA38 has 2 PMTs and one 32 channel PMT array. The PMT array was engineered to enable the separation of fluorophores such as, Alexa Fluor 532 and BV57. The separation of such overlapping combinations is further enhanced through spectral unmixing software.
Application Data, SA38 Spectral Analyzer TBNK Panel Page 2 Normal human PBMCs were stained with the TBNK panel and analyzed on the SA38. B and T-Cells CD4 and CD8 T-Cells A B C D All events : 94.68% x1, 3 CD19+: 12.74% CD8+: 26.26% 1 SSC_A 1 : 21.26% CD19_BV65_A CD8_BV57_A CD4+: 66.59% - 5 1 FSC_A x1, - - CD45_Alexa Fluor 582_A - - - : 72.58% - - - CD4_PE-AF7_A E F G H 3 x1, 3 CD3- CD7+CD16/56++: 43.82% CD4+ CD7+CD16/CD56++: 18.89% Monocyte: 7.7% SSC_A CD7 A CD7-CD16/56--: 49.36% CD7 A 1 1 CD3: 26.15% - - - - - - CD7-CD16/56++: 5.8% - - CD14 A CD16/CD56 A CD16/CD56 A Monocytes NK Cells (Target Cell Engagers) CD8+ Cytotoxic T Cells TBNK data analyzed with traditional dot plots. Forward and side scatter combined with CD45 was used to identify lymphocytes (B), from the lymphocyte population B and T cells were identified (C). The T-cell population was further divided into CD4 and CD8 T-cells (D). From the CD8 T-cell population cytotoxic T-cells were identified using CD7 and CD16/CD56 (H). From the forward and side scatter profiles with the addition CD14 (E) the percentage of monocytes was determined. To examine the NK cells lymphocytes (B) CD3 was used to remove the T-cells (F). Figure G shows the population of NK cells.
Application Data, SA38 Spectral Analyzer TBNK Panel Page 3 Spectral plots represent another method to examine the same data set. When viewing single color fluorochrome controls, peaks within the fluorochrome signature are easily identified. BV57 BV65 BV57 BV65 45nm 45nm AF532 PE-AF7 AF532 PE-AF7 488nm 488nm 638nm 638nm Plots highlight the individual fluorochromes within the spectra. Arrows in the plots on the right highlight the fluorochrome peak and its corresponding wavelength. s correspond to the expected emission peaks.
Application Data, SA38 Spectral Analyzer TBNK Panel Page 4 SSC_A x1, 3 1 : 21.26% CD19_BV65_A FMO s phocytes CD19+: 13.3% - - :.% - - CD19_BV65_A - phocytes CD19+: 12.78% CD19-:.% - : 72.54% - - CD45_Alexa Fluor 532_A B and T-Cells phocytes CD19+: 12.74% CD19_BV65_A - - - - CD3-: 74.66% - - CD8+: 26.28% CD19_BV65_A CD8_BV57_A CD4+: 66.57% - CD19+ BV65 - - - : 72.58% - - - CD4_PE-AF7_A CD19+ CD19+ BV65 CD8+ CD4+ CD4+ PEAF7 LD488_A CD8+ BV57 LD488_A 5 6 7 8 5 6 7 8 BV65 CD19+ B Cells T Cells Dye Specificity Purpose Dye Specificity Purpose AF532 CD45 phocytes BV57 CD8 Suppressor/Cytotoxic T phocytes PE-Alexa Fluor 7 CD4 Helper/Inducer T phocytes CD3 T phocytes BV65 CD19 B phocytes B-cell and T-cell populations (stained with BV57 and AF532 respectively) are identified through conventional bivariant dot plots. Populations can also be identified by their location in the spectral plot. CD19+ cell populations are represented by the peak at 65nm while T-cells can be seen at the 53 nm peak. CD4 and CD8 T-cells stained with PE-AF7 and BV57 respectively. The CD4 spectral plot (stained with PE-AF7 ) shows a peak at 7nm for CD4 + cells. The CD8 spectral plot (stained with BV57) shows a peak on the x-axis for CD8+ cells. To improve accuracy, SA38 software can also be used to verify dot plot gating simply by moving the dot plot gate while observing the excitation peaks in the spectral plot and adjusting the dot plot gate accordingly. Spectral plots can also be gated for deeper analysis, if needed. Conclusion Spectral analysis is a new method to examine cell populations. Results of experiments with spectral analysis are comparable or better to that of conventional flow cytometry with some key advantages. Richer data sets can be generated facilitating easy identification of tandem degradation, sample degradation, and staining abnormalities. In addition, with spectral analysis many fluorochromes with overlapping spectra, such as and AF532, can be used together enabling the use of more fluorochromes off the same laser. This allows more data to be generated from limited samples and more flexibility in experimental design.compromising data quality. 1 Milush, Jeffrey M., et al. CD56negCD16+ NK cells are activated mature NK cells with impaired effector function during HIV-1 infection. Retrovirology 1.1 (13): 158.
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