Modern developments in flow cytometry, combined with the ongoing emergence of novel fluorescent labels have enabled an unprecedented level of analysis in cell biology, immunology, and microbiology, as well as, cancer and clinical research studies. With these tools, distinct populations of cells can be screened and characterized on a cell-by-cell basis for specific, biologically-significant traits, including cell surface markers and secreted markers, intracellular targets, enzyme activity, nucleic acid content, apoptosis and much more. AAT Bioquest provides a comprehensive portfolio of flow cytometry reagents ranging from fluorescent labels and CD antibody conjugates to fixable viability dyes, cell proliferation dyes and tools to monitor apoptosis.
Super Bright Fluorescent Dyes, Phycobiliproteins and Tandem Dyes for Multicolor Flow Cytometry
Flow cytometry analysis of human PBMC cells. Anti-human CD4 antibody prepared with Buccutite™ Rapid PE Antibody Labeling Kit (Cat No. 1310) or Lightning-Link® PE. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the FITC channel.
Fluorescent dyes conjugated to antibodies or proteins enable biological and biochemical properties to be measured in a wide range of flow cytometric applications. Across most experimental conditions, phycobiliproteins such as PE and APC
are among the brightest fluorophores available for flow cytometry. While novel mFluor™ dyes
offer users greater photostability, superior brightness and exceptionally long Stoke's Shifts for multiparametric analysis.
To label primary antibodies or proteins for flow cytometry, AAT Bioquest offers a wide selection of antibody labeling kits. Our ReadiLink™ Rapid mFluor™ Antibody Labeling Kits
and Buccutite™ PE, APC and Tandem dye Antibody Labeling Kits
, enables researchers to effortlessly label and recover 100% of their antibodies without a purification step. Produce stable covalently-labeled conjugates for FACS, immunophenotyping, multiplex flow cytometry and more.
Table 1. Buccutite™ Rapid Antibody Labeling Kits
|Buccutite™ Rapid Protein Crosslinking Kit *Microscale Optimized for Crosslinking 100 ug Antibody Per Reaction*||-||-||2 Labelings||1315|
|Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 1 mg Protein*||-||-||1 Kit||5504|
|Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 100 ug Protein*||-||-||1 Kit||5503|
|Buccutite™ Peroxidase (HRP) Antibody Conjugation Kit *Optimized for Labeling 25 ug Protein*||-||-||2 Labelings||5505|
|Buccutite™ Rapid PE Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*||565||575||2 Labelings||1310|
|Buccutite™ Rapid PE Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*||565||575||2 Labelings||1312|
|Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*||565||600||2 Labelings||1318|
|Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*||565||600||2 Labelings||1343|
|Buccutite™ Rapid PE-Cy5 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*||565||674||2 Labelings||1322|
|Buccutite™ Rapid PE-Cy5 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 25 ug Antibody Per Reaction*||565||674||2 Labelings||1340|
Cell Proliferation Reagents for Flow Cytometry
Cell proliferation assays and reagents are important tools used in cell growth and differentiation studies to monitor the health of cells within a heterogeneous population. They have been used extensively in pharmaceutical drug development to examine compound toxicity and inhibition of tumor cell growth, and have proved to be a successful diagnostic tool to detect the onset of several cancers. In flow cytometry, several approaches can be used to measure proliferation; these include dye dilution assays and DNA synthesis measurements through the incorporation of Bromodeoxyuridine (BrdU)
Dye dilution assays for cell proliferation
CFSE and CytoTell™ proliferation dyes can be used in flow cytometry to examine cell proliferation in living cells. As cells divide, the fluorescent dye is uniformly distributed between daughter cells, resulting in the progressive halving of fluorescence intensity with each successive generation. These proliferation dyes can be used to track cell proliferation long-term both in vitro
and in vivo
for several generations.
CFSE (Carboxyfluorescein diacetate, succinimidyl ester) is a cell-permeable green fluorescent proliferation indicator that emits a fluorescence signal at 517 nm when excited at 498 nm. This dye passively diffuses across uncompromised cell membranes, whereby enzyme reactions with intracellular esterases cleave acetate groups of CFSE to yield an amine-reactive carboxyfluorescein, SE that covalently binds to proteins within the cell. While CFSE still remains widely used as a green fluorescent proliferation indicator excited at 498 nm it is, however, not without caveats. CFSE is highly toxic to cells, susceptible to dye leakage from the cell and is known to spillover into the PE and PE-Texas Red channels.
High-performance CFSE upgrades for cell proliferation
Cell proliferation assay using CytoTell™ Green and CFSE. Jurkat cells (~2x106 cells/mL) were stained with CytoTell™ Green or CFSE on Day 0. Cells were then passed serially at 1:1 ratio on the day specified. Fluorescence intensity was measured with ACEA NovoCyte 3000 flow cytometer in FITC channel on the day of passage. Successive generations are represented by different color peaks.
CytoTell™ are a series of cell-permeable fluorecent tracers designed to monitor cell proliferation in living cells without any of the drawbacks associated with CFSE. Upon passive diffusion across cell membranes, CytoTell™ dyes are hydrolyzed by intracellular esterases to yield highly-fluorescent amine-reactive or thiol-reactive dyes that covalently bind to amine or thiol groups on intracellular proteins. Cytotoxicty is minimal and the dyes are well-retained within cells through several generations of division, up to 9 generations can be visualized using CytoTell™ UltraGreen. CytoTell™ dyes are available in 7 fluorescent stains offering greater flexibilty in multicolor analysis, and may be fixed and permeabilized for analysis of intracellular targets using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers.
Table 2. CytoTell™ and CFSE cell proliferation dyes for flow cytomtery
|CytoTell™ Blue||Violet Laser (405 nm)||410||445||500 Tests||22251|
|CytoTell™ Violet 500||Violet Laser (405 nm)||415||499||500 Tests||22248|
|CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]||Blue Laser (488 nm)||498||517||25 mg||22022|
|ReadiUse™ CFSE [5-(and 6)-Carboxyfluorescein diacetate, succinimidyl ester]||Blue Laser (488 nm)||498||517||5 x 500 µg||22028|
|CytoTell™ Green||Blue Laser (488 nm)||510||525||500 Tests||22253|
|CytoTell™ UltraGreen||Blue Laser (488 nm)||492||519||500 Tests||22240|
|CytoTell™ Orange||Green Laser (531 nm)||541||560||500 Tests||22257|
|CytoTell™ Red 590||Green Laser (531 nm)||560||574||500 Tests||22261|
|CytoTell™ Red 650||Red Laser (633/647 nm)||626||643||500 Tests||22255|
|CytoTell™ Blue||Violet Laser (405 nm)||410||445||2 x 500 Tests||22252|
Fixable Viability Dyes for Flow Cytometry
Cell Meter™ staining principle. Viable cells will exclude Cell Meter™ fixable viability dyes leaving only cell surface amines and thiols to bind with, resulting in minimal fluorescence. Dead cells with compromised membrane integrity will allow the dye to penetrate and stain amine- and thiol-containing proteins and other intracellular components resulting in intense fluorescence. Difference in signal intensity between viable and dead cells is significant, making it easy to exclude dead cells from analysis.
The ability to discriminate and exclude dead cells from live cells enables cleaner separation and identification of cell populations. It greatly enhances assay accuracy, and eliminates undesirable artifacts and false positives caused by the nonspecific binding of various reagents to dead cells. To distinguish live and dead cell populations from flow cytometry readouts AAT Bioquest offers a broad range of Cell Meter™ Fixable Viability dyes and Live or Dead™
Fixable Dead Cell Staining Kits. Labeling is stable, robust and compatible with fixatives and permeabilization buffers allowing for further analysis of intracellular targets.
Cell Meter™ fixable viability dyes
Detection of Jurkat cell viability by Cell Meter™ fixable viability dye. Jurkat cells were treated and stained with Cell Meter™ BX590 (Cat#22514), and then fixed in 3.7% formaldehyde and analyzed by flow cytometry. The dead cell population (Blue peak) is easily distinguished from the live cell population (Red peak) with PE-TexasRed channel, and nearly identical results were obtained before and after fixation.
Cell Meter™ fixable viability dyes are a family of membrane-impermeant viability dyes designed to distinguish live and dead cells during flow cytometry. These dyes readily permeate dead cells with compromised membrane integrity and bind irreversibly to amine- and thiol-containing proteins and other intracellular components making it easy to exclude them from analysis. Because staining is robust and stable, cells labeled with Cell Meter™ fixable viability dyes can be fixed, permeabilized and probed for intracellular immunophenotyping.
The Cell Meter™ fixable viability dyes are available in 9 fluorescent stains to provide users greater flexibility in multicolor panel design. Dyes included in this series are excited by major excitation sources including the 405, 488, 633 and 647 nm laser lines, and emit in common channels.
Table 3. Fixable viablity dyes for live/dead cell analysis during flow cytometry
|Cell Meter™ VX450 fixable viability dye||406||445||200 Tests||22540|
|Cell Meter™ VX500 fixable viability dye||433||498||200 Tests||22542|
|Cell Meter™ BX520 fixable viability dye||491||516||200 Tests||22510|
|Cell Meter™ BX590 fixable viability dye||492||579||200 Tests||22514|
|Cell Meter™ BX650 fixable viability dye||518||654||200 Tests||22520|
|Cell Meter™ RX660 fixable viability dye||649||664||200 Tests||22530|
|Cell Meter™ RX700 fixable viability dye||690||713||200 Tests||22532|
|Cell Meter™ RX780 fixable viability dye||629||767||200 Tests||22536|
|Cell Meter™ IX830 fixable viability dye||811||822||200 Tests||22529|
Live or Dead™ Fixable Dead Cell Staining Kits
Live or Dead™ Fixable Dead Cell Staining Kits employ membrane-impermeant amine-reactive dyes to differentiate live and dead cells during flow cytometry. The dyes provided in each kit can readily permeate compromised membranes of dead cells and covalently bind to both intracellular and extracellular amine-containing proteins resulting in intense fluorescence. While on live cells, the fluorescence signal is negligible as cell surface amine-groups are only avaiable to react with the dye. The difference in signal intensity between live and dead cell populations is signifcant, making it easy to exclude dead cells from analysis. Cells labeled using Live or Dead™ Fixable Dead Cell Staining Kits can be fixed, permeabilized and probed for intracellular immunophenotyping. AAT Bioquest offers 10 Live or Dead™ Fixable Dead Cell Staining Kits designed to for efficient excitation by major exciation sources including the 350, 405, 488, 532, 561, 633 and 647 nm laser lines, and emit in common channels.
Detection of Jurkat cell viability by Live or Dead™ Fixable Dead Cell Staining Kits. Jurkat cells were treated, stained and then fixed in 3.7% formaldehyde and analyzed by flow cytometry.
Table 4. Live or Dead™ Fixable Dead Cell Staining kits for live/dead cell analysis during flow cytometry
|Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence*||UV (350 nm)||353/442||Yes||Yes||200 Tests||22600|
|Live or Dead™ Fixable Dead Cell Staining Kit *Blue Fluorescence with 405 nm Excitation*||Violet (405 nm)||410/450||Yes||Yes||200 Tests||22500|
|Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence with 405 nm Excitation*||Violet (405 nm)||408/512||Yes||Yes||200 Tests||22501|
|Live or Dead™ Fixable Dead Cell Staining Kit *Green Fluorescence*||Blue (488 nm)||498/521||Yes||Yes||200 Tests||22601|
|Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence with 405 nm Excitation*||Violet (405 nm)||398/550||Yes||Yes||200 Tests||22502|
|Live or Dead™ Fixable Dead Cell Staining Kit *Orange Fluorescence*||Green/Yellow (532/561 nm)||547/573||Yes||Yes||200 Tests||22602|
|Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence*||Yellow (561 nm)||583/603||Yes||Yes||200 Tests||22603|
|Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*||Blue/Green (488/532 nm)||523/617||Yes||Yes||200 Tests||22599|
|Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*||Red (635 nm)||649/660||Yes||Yes||200 Tests||22604|
|Live or Dead™ Fixable Dead Cell Staining Kit *Red Fluorescence Optimized for Flow Cytometry*||Red (635 nm)||749/775||Yes||Yes||200 Tests||22605|
Live and Fixed Cell Cycle Assays for Flow Cytometry
DNA profile in growing and nocodazole treated Jurkat cells. Jurkat cells were treated without (A) or with 100 ng/ml Nocodazole (B) in a 37 °C, 5% CO2 incubator for 24 hours, and then dye loaded with Nuclear Green™ LCS1 for 30 minutes. The fluorescence intensity of Nuclear Green™ LCS1 was measured with ACEA NovoCyte flow cytometer with the channel of FITC. In growing Jurkat cells (A), nuclear stained with Nuclear Green™ LCS1 shows G1, S, and G2 phases. In Nocodazole treated G2 arrested cells (B), frequency of G2 cells increased dramatically and G1, S phase frequency decreased significantly.
Analyzing changes in DNA content during flow cytometry is a powerful method commonly used to identify cells in different stages of the cell cycle. This technique utilizes fluorescent DNA binding dyes, which bind to DNA stoichiometrically, generating a fluorescence signal that is directly proportional to the amount of DNA present in the cell. This way when the DNA density increases as cells progress from the G1 phase to the G2 phase of the cell cycle, cells will take up proportionally until the cell have doubled their DNA content. As a result cells in the G2 phase will fluoresce twice as brightly as cells in the G1 phase.
AAT Bioquest offers a selection of 4 cell cycle assays, 3 of which are designed for live cell analysis and 1 for fixed cells. The Cell Meter™ Fluorimetric Live Cell Cycle Assay Kits utilize membrane-permeant DNA binding dyes to monitor cell cycle progression and proliferation, and can be used to investigate the kinetic and temporal aspects of the cell cycle. After staining cells can be fixed and permeabilized. The Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit utilizes our proprietary membrane-impermeant DNA binding dye Nuclear Red™ CCS1. Prior to staining, cells must be fixed or permeabilized to all the dye to enter the cell. Each kit includes a robust staining protocol and all the necessary components for 200 tests. AAT Bioquest also offers a large selection of stand-alone DNA binding dyes for cell cycle analysis, including propidium iodide
, Hoechst 33342
, and TWO-PRO™ 1
and TWO-PRO™ 3
Table 5. Live and fixed cell cycle assays for flow cytometry
|Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Optimized for 405 nm Violet Laser Excitation*||Violet Laser (405 nm)||401||460||100 Tests||22845|
|Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Green Fluorescence Optimized for Flow Cytometry*||Blue Laser (488 nm)||503||527||100 Tests||22841|
|Cell Meter™ Fluorimetric Live Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*||Blue Laser (488 nm)||488||615||100 Tests||22860|
|Cell Meter™ Fluorimetric Fixed Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*||Blue/Green Laser (488/532 nm)||537||682||100 Tests||22842|
Apoptosis Assay for Flow Cytometry
Flow cytometry is one of the most versatile and high-throughput applications for studying the complexities of apoptosis. Not only does it enable researchers to analyze cells individually from complex populations, but the multiparametric nature of flow cytometry allows for several apoptotic indicators and markers to be examined simultaneoulsy. With our broad selection of flow cytometric apoptosis assays investigate changes in plasma membrane conformation, loss of mitochondrial membrane potential, activation of caspases, nuclear condensation or utilize a combination of techniques for multiparametric apoptosis analysis.
Annexin V assays for the detection of apoptosis by flow cytometry
Annexin V conjugates are routinely used to investigate the externalization of phosphatidylserine (PS) residues, a key characteristic in early-stage apoptosis. As cells initiate programmed cell death, they lose plasma membrane asymmetry and PS residues translocate from the inner to the outer leaflet of the plasma membrane. Exposure of PS residues on the cell surface can be detected by its affinity for annexin V, and when fluorescently labeled annexin V conjugates can be used to identify apoptotic cells in flow cytometry.
AAT Bioquest Cell Meter™ Annexin V Binding Apoptosis Assay Kits offer a quick and reliable method for measuring apoptotic cells based on the externalization of PS residues. These kits utilize a highly fluorescent annexin V conjugate and a membrane-impermeant DNA stain to differentiate between live, dead and apoptotic cells. In this way, cells that are viable show little or no fluorescence, cells undergoing early apoptosis show annexin V conjugate fluorescence and dead cells show a double labeling of the membrane-impermeant DNA stain and annexin V conjugate. These kits can be paired with other reagents for multi-parametric study of cell viability and apoptosis.
Key features of Cell Meter™ Annexin V Bindinng Apoptosis Assays
The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 µM staurosporine (Red) in 37°C for 4 hours, and then ed with Annexin V-mFluor™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in Pacific Blue channel.
- High Sensitivity - annexin V labeled with iFluor® or mFluor™ dyes produce significantly brighter and more photostable signals than Alexa Fluor® and other spectrally similar annexin V conjugates
- Flexibility - wide selection of stand-alone annexin V conjugates or easy-to-use assay kits to match any instrument set-up
- Convenient assay design - streamlined protocol that can be completed in 30 to 60 minutes
Table 6. Cell Meter™ Annexin V Binding Apoptosis Assay Selection Guide
|Annexin V-iFluor® 488||PI||490/ 520 nm||534/617 nm||100 Tests||22824|
|Annexin V-iFluor® 555||None||554/578 nm||***||100 Tests||22825|
|Annexin V-iFluor® 594||None||590/610 nm||***||100 Tests||22826|
|Annexin V-iFluor® 647||None||650/668 nm||***||100 Tests||22827|
|Annexin V-mFluor™ Violet 450||PI||405/450 nm||534/617 nm||100 Tests||22828|
|Annexin V-mFluor™ Violet 500||PI||414/508 nm||534/617 nm||100 Tests||22829|
|Annexin V-mFluor™ Violet 550||PI||424/560 nm||534/617 nm||100 Tests||22830|
|Annexin V-APC||PI||651/662 nm||534/617 nm||100 Tests||22837|
|Annexin V-PE||Nuclear Red™ DCS1||565/575 nm||631/651 nm||100 Tests||22838|
|Annexin V-FITC||PI||490/520 nm||534/617 nm||100 Tests||22839|
- For Cat No. 22825, 22826 and 22827 use Nuclear Blue™ DCS1 as a dead cell stain.
Mitochondria membrane potential assays for flow cytometry
Loss of mitochondrial membrane potential is a distinctive feature of the early stages of apoptosis. In flow cytometry, membrane-permeant cationic probes can be used to identify changes in the mitochondrial membrane potential in real-time and discriminate apoptotic cells from live cell populations. AAT Bioquest offers a wide selection of reagents and dynamic assays designed to monitor mitochondrial membrane potential, these include ratiometric indicators such as JC-10™, single-emission indicators, and Cell Meter™ Mitochondrion Membrane Potential Assay Kits optimized for flow cytometry applications.
Effect of FCCP induced mitochondria membrane potential change in Jurkat cells. Jurkat cells were dye loaded with JC-10 dye working solution along with DMSO (Top) or 5 µM FCCP (Low) for 10 minutes. The fluorescence intensities for both J-aggregates and monomeric forms of JC-10 were measured with a FACSCalibur (Becton Dickinson) flow cytometer using FL1 and FL2 channels. Uncompensated data (left column) were compared with compensated data (right column).
AAT Bioquest JC-10™ is a novel ratiometric indicator designed to monitor mitochondrial membrane potential with greater accuracy and sensitivity than its predecessor JC-1
. Similar to JC-1, JC-10™ exhibits potential dependent accumulation. In healthy cells, JC-10™ selectively accumulates in mitochondria generating orange J-aggregates that exhibit a broad excitation spectrum and emission maximum at 590 nm. Conversely, in apoptotic and necrotic cells with low mitochondrial membrane potential, JC-10™ diffuses out of the mitochondria and JC-10™ monomers are generated, resulting in a shift to green emission (525 nm). JC-10™ allows both qualitative and quantitative visualization of mitochondrial membrane potential changes, considering the shift from orange to green fluorescence emission and the fluorescence intensity ratio, respectively. JC-10™ dye is available as a standalone reagent or in the Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*
Key features of JC-10™ and the Cell Meter™ JC-10™ assay kit
The decrease in fluorescence intensity of MitoTell™ Red in response to CCCP treatment in Jurkat cells. Jurkat cells were loaded with MitoTell™ Red alone (Blue) or in the presence of 20 µM (Green) or 50 µM CCCP (Red) for 30 minutes. The fluorescence intensity of MitoTell™ Red was measured using ACEA NovoCyte flow cytometer at APC channel.
- Easy-to-Use: JC-10™ does not percipitate when diluted into aqueous buffers, eliminating artifacts.
- Robust: JC-10™ has smaller assay deviations due to its enhanced solubility in aqueous media and higher sensitivity.
- Enhanced Signal: JC-10™ has a higher signal-to-background ratio than JC-1.
- Enhanced Sensitivity: JC-10™ has the ability to detect subtle changes in ΔΨm loss better than JC-1 in all tested cell lines.
Single-emission mitochondrial membrane potential dyes
Other reagents and assays that can be used to dynamically monitor changes in mitochondria membrane potential include TMRE, TMRM and the Cell Meter™ Mitochondrion Membrane Potential Assay Kits available in orange, red and near-infrared fluorsecence. In healthy cells with functioning mitochondria, these membrane-permeant dyes are readily sequestered by active mitochondria and fluoresce brightly. When mitochondria membrane potential collapses, accumulation ceases and the dyes are released throughout the cytosol resulting in loss of fluorescence.
Table 7. Reagents and assays for monitoring mitochondrial membrane potential during flow cytometry
|JC-1 ||Blue Laser (488 nm)||515||530||5 mg||22200|
|JC-1 ||Blue Laser (488 nm)||515||530||50 mg||22201|
|JC-10™||Blue Laser (488 nm)||508||524||50 mg||22201|
|TMRE||Green Laser (532 nm)||552||574||25 mg||22220|
|TMRM||Green Laser (532 nm)||552||574||25 mg||22221|
|Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*||Blue Laser (488 nm)||488||530, 575 nm||100 Tests||22801|
|Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*||Blue/Green Laser (488/532 nm)||540||590||100 Tests||22804|
|Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Flow Cytometry*||Red Laser (640 nm)||640||660||100 Tests||22806|
|Cell Meter™ NIR Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry*||Red Laser (633-640 nm)||635||660||100 Tests||22806|