SP6800 Spectral Analyzer

16 color panel on Human Peripheral Blood

Application data using 2 lasers

Fluorochromes: 488nm Laser

human-peripheral-2L-488nm

CD3 Alexa Fluor 488
CD8 PE
CD4 PE Dazzle 594
CD24 PE Alexa Fluor 610
CD19 PE Cy5
CD20 PE Cy5.5
CD13 PerCP efluor® 710
CD45 PE Cy7

Fluorochromes: 405nm Laser

human-peripheral-3L-405nm

HLA-DR Pacific Blue
CD38 BV421
CD45RA BV510
CD33 BV570
CD16 BV605
CD11b BV650
CD11c BV711
CD14 BV785

human-peripheral-2L-16-color

Examples of the resolution of several important cell populations using only two lasers with the SP6800. Clear population resolution is obtained with highly overlapping fluorochomes such as PE-Cy5/PE-Cy5.5 (G) and Pacific Blue/BV421 (H).

16 color stained sample of Human Peripheral Blood

Application data using 3 lasers

Fluorochromes: 405nm Laser

human-peripheral-3L-405nm

HLA-DR Pacific Blue
CD5 BV421
CD56 BV510
CD33 BV570
CD16 BV605

Fluorochromes: 488nm Laser

human-peripheral-2L-488nm

CD3 Alexa Fluor 488
CD8 PE
CD4 PE Dazzle 594
CD24 PE Alexa Fluor 610
CD19 PE Cy5
CD20 PE Cy5.5
CD13 PerCP efluor® 710
CD45 PE Cy7

Fluorochromes: 638nm Laser

human-peripheral-3L-638nm

CD38 APC
C11c Alexa Fluor 700
CD45RA APC-Cy7

human-peripheral-3L-16-color

Example of the detection of several important cell populations using sixteen fluorochromes excited by three lasers. Unlike conventional flow cytometers that can detect up to five off the blue, in this example we demonstrate that with the SP6800 eight fluorochromes can be excited off the blue with clear sample resolution. Even highly overlapping fluorchromes such as PE-Cy5 and PE-Cy5.5 can be resolved.

Application Data: Brilliant Violet Dyes

Spectral Analysis allows multi-laser excited fluorochromes to be run without using a complicated compensation matrix.

human-peripheral-2L-405nm

BV421 BV605
BV510 BV650
BV570 BV711
BV785

brilliant-human-pbmc-graphs

Sample data from Human PBMCs stained with seven markers conjugated to Brilliant Violet dyes offered by Sony Biotechnology. D. All populations were clearly resolved including fluorochomes with significant spectral overlap such as BV605 and BV650 (D).

12-color Staining of Human Peripheral Blood Leukocytes

Example of Human Peripheral Blood Leukocytes was analyzed on the SP6800 and a BD LSRFortessa conventional flow cytometer. Spectral analysis was able to remove the autofluorescenece and use unmixing to separate each fluorochrome which resulted in clarity and resolution of each population in these density plots.
12-color-leukocytes-table
12-color-leukocytes

12-color-leukocytes-slide

Example 1

12-color-leukocytes-slide

Example 2

12-color-leukocytes-slide

Example 3

12-color-leukocytes-slide

Example 4

12-color-leukocytes-slide

Example 5

Application Data: Dyes with issues

Common problems in flow cytometry occur when running fluorochromes with emission peaks that are too close to one another, multi-laser excitations, fluorescent proteins, and unstable tandems. The SP6800 is capable of analyzing all of these by looking at all photons from 420nm to 800nm and unmixing each unique spectral fingerprint.

6800-applications-issues

Example 1: Pacific Blue (457nm emission) and Brilliant Violet 421 (422nm emission)

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Example 2: PE Cy5 (excited with 488nm and 638nm lasers) and APC (excited with 638nm laser)

6800-applications-issues

Example 3: Fluorescent GFP and YFP need no special bandpass filter set with the SP6800

6800-applications-issues

Example 4: PE Cy7 tandem is prone to falling apart causing issues with resolution when using conventional flow cytometers. Since Spectral Analysis unmixes the spectral fingerprint, the SP6800 produces excellent resolution.