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Keywords:

  • cell analysis;
  • Immunophenotyping;
  • violet-excitable dye;
  • multicolor FCM;
  • lymphocytes;
  • subsetting

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

We have recently added three new fluorophores–BD Horizon™ V450, BD Horizon V500, and BD Horizon V550 (V450, V500, and V550; BD Biosciences, San Jose, CA) to our existing AmCyan product, forming a group of four violet-excitable dyes from which we have produced functional antibody conjugates. These conjugates, with emission maxima that range from 450 to 535 nm, are compatible with multilaser flow cytometry (FCM) and can be used for polychromatic FCM in three-color or two-color combinations; in fact, V500 fills a spectral opening that has thus far not been exploited by other manufacturers of FCM reagents. We here report that conjugates based on BD Horizon dyes performed well within a useful sensitivity range, established by testing a representative group of violet-excitable FCM reagents currently available, and that V500 has better compatibility with FITC in multicolor applications than does AmCyan. © 2013 International Society for Advancement of Cytometry

Multiparameter FCM and cell sorting have become essential tools for phenotypic and functional cellular measurements over the last two decades ([1-3]). The availability of new lasers has encouraged the development of additional fluorochromes, thereby increasing the number of parameters that can be probed simultaneously. We believe that upcoming advances in LED technology will make high quality, stable violet sources available at low cost in the very near future. Consequently, the availability of new water-soluble violet-excitable dyes designed for conjugation to antibodies will continue to expand significantly in the next few years.

AmCyan, a violet-excitable dye first introduced in 2004, had a suitable emission profile for use as a second violet dye with Cascade® Blue, Alexa Fluor® 405, or Pacific Blue™, which were in widespread use at the time. On the basis of a very low absorption at 488 nm (less than 1% of peak value), AmCyan also appeared to have sufficiently low blue laser excitation so as not to require application of significant compensation to the fluorescein-assigned (FITC) detector (525/50; 488 nm laser). This suggested that it would be practical to use AmCyan in multicolor panels also containing FITC. However, in practice, significant compensation was required when this combination was used, seriously compromising the sensitivity of FITC reagents. Because our previous introduction of the violet-excitable dye V450 ([4]) was successful, we felt confident in pursuing an organic dye with AmCyan-like emission characteristics without the aforementioned deficiencies. Therefore, the next objective was to either improve the excitation characteristics of AmCyan or to develop a new dye with an emission maximum of around 500 nm and little or no excitation by the blue laser. We decided to synthesize a new dye with green (500 nm) emission because initial results suggested it would be more efficient. When a suitable candidate was identified, we named this coumarin dye V500, and here report various evaluations and characterizations, including comparisons with AmCyan. This pursuit also led to the synthesis and evaluation of other dye candidates, some of which were oxazole derivatives with orange emission. Although oxazoles (e.g., Pacific Orange™) have typically been among the least desirable violet-excitable dyes used in FCM because of their inferior brightness and their rather broad emission profiles, we felt that one of these candidates warranted further investigation. This particular orange-emitting dye (referred to as V550) has longer Stokes shifts than coumarin dyes (such as V450), and appears to be brighter than other oxazoles. Preliminary characterization and conjugation results are included in this report.

In this report, we assess spectral properties, brightness, results of conjugation to monoclonal antibodies, and performance in FCM of a broad collection of violet-excitable dyes including V450, V500, V550, and AmCyan. We also introduce a novel conjugation chemistry (V500-C), which mitigates compensation issues reported ([5]) when using BD™ CompBeads (BD Biosciences, San Jose, CA) and demonstrate the use of CD3 V500-C in multicolor staining assays designed to study either the phosphorylation of intracellular signaling proteins or T cell memory.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Absorption and Emission Spectra

Succinimidyl esters of thiophenyl-substituted hydroxycoumarins were diluted in sodium bicarbonate buffer at pH 9.6 and spectra were measured in 1 cm cuvettes on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA).

Molar Extinction Coefficients

The molar extinction coefficient (ε) of a chemical species is a measure of its ability to absorb light of a specified wavelength. Molar extinction coefficients for V450, V500, and V550 were determined as follows: weighed samples of the dyes were dissolved in measured volumes of dimethylsulfoxide (DMSO) to obtain concentrated stock solutions of known concentrations (mg/ml; C). These solutions were then diluted appropriately for spectrophotometry by a dilution factor (D) in sodium bicarbonate pH 9.5 and absorption spectra were then recorded in 1 cm cuvettes on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA). Maximum absorbances were determined (Amax) from the absorption spectra and molecular weights (W) were derived based on the structures (Fig. 1 and Ref. [4]).

image

Figure 1. Structures of BD Horizon™ V450, V500, and V550. The structures are used to calculate the molecular weights necessary for the derivation of molar extinction coefficients and illustrate the presence of a thiophenyl group on V500 that is responsible for shifting the emission spectrum into the green region.

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ε for each dye was then calculated from the following equation:

  • display math

Fluorescence Quantum Yield

Fluorescence quantum yields (QY) of glycinated V500 or V550 were determined using either quinine sulfate (Sigma-Aldrich, St. Louis, MO) or V450 as reference standards, and values of 0.51 ([6]) or 0.98, respectively, for quantum efficiency ([4]). All readings were taken at ambient temperature. V450-, V500-, and V550-glycine were serially diluted in 5 mM sodium phosphate buffers with A375 or A400 ≤ 0.1, and quinine sulfate was serially diluted (with A375 ≤ 0.1) in 50 mM sulfuric acid. Absorbance of all dyes was measured in 1 cm cuvettes on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies). Corrected emission spectra were recorded on a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ) in 1 cm cuvettes using excitation at 375 nm for all dyes except V550, which was done in 96-well plates on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) using excitation at 400 nm. Data were plotted as integrated fluorescence intensities (IFI) versus absorbance, and were analyzed using Microcal Origin software. QYs were calculated using the equation: inline imagewhere Q is the quantum yield, n is the refractive index, and Grad is the gradient (slope) obtained from the plot of the IFI vs. absorbance. The subscript R refers to the reference fluorophore of known QY.

Effect of pH on Dye Fluorescence

V450 was dissolved to a final concentration of 1 µM in a series of buffers at different pHs (acetate buffers were used in the pH range of 4–6, and phosphate buffers in the pH range of 6–8). Samples of V500 over a range of pHs were also prepared at equal dilutions from a common stock solution to an approximate final concentration of 5 µM in appropriate buffers (50 mM potassium phosphate for pH 3 to 8.5 and 50 mM sodium bicarbonate for pH 9–10). Fluorescence emission determinations were then made at close intervals (e.g., 1 pH unit or less from pH 3 to 6, and 0.5 pH units or less from 6.5 to 10). Measurements for both V450 and V500 were done in a SpectraMax Gemini fluorescent microplate reader (Molecular Devices, Sunnyvale, CA) at a volume of 100 µl per well in a 96-well black solid plate using excitation at 405 nm and emission detection at 460 or 500 nm, respectively. Samples of V550 were similarly prepared over a pH range from 5.0 to 9.5 and to an approximate final concentration of 10 µM in appropriate buffers (50 mM sodium citrate for pH 5, 50 mM sodium phosphate for pH 6–8.15 and 50 mM sodium bicarbonate for pH 9.5). V550 samples were then analyzed for fluorescence emission with a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ) in 1 cm cuvettes using excitation at 405 nm and emission detection at 535 nm.

Conjugations

Monoclonal antibody was buffer-exchanged into labeling buffer on spin columns (Bio-Spin 30, Bio-Rad Laboratories, Hercules, CA) and concentrated to 2–20 mg/ml on Vivaspin 50 kDa MWCO or equivalent centrifugal concentrators, as necessary. NHS esters of V450, V500, or V550 dyes were dissolved in DMSO and their concentrations were determined spectrophotometrically. The dyes were diluted into pH 9.6 labeling buffer before reading. Labeling was done for 60 min at ambient temperature. A portion of each reaction mixture was separated on a Bio-Spin column and analyzed for dye-to-protein ratio (D/P) and antibody concentration spectrophotometrically. The samples were then tested by lyse-wash staining at saturating concentrations of antibody conjugate as described below.

Some conjugates (V500-C, BD Biosciences proprietary technology) were prepared by first labeling a 17,000 Da protein with V500 and succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and then conjugating the resulting modified fluorophore to antibody sulfhydryl groups followed by purification on gel filtration columns. Both V500-C and V500 conjugates were used in the 15-reagent comparison experiment (Fig. 5). V500-C (and not directly labeled V500 conjugates) were used in seven and eight-color experiments (Figs. 9 and 10). V500 directly labeled conjugates (and not V500-C) were used in all other experiments.

Flow Cytometry

Antibody clones

All experiments involving CD3, CD8, or CD45 reagents were made from clones SK7, SK1, or 2D1, respectively, and CD4 reagents used in all experiments except those in Figure 5 were from clone SK3 (BD Biosciences, San Jose).

The sources and clones of CD4 reagents shown in Fig. 5 are as follows: Pacific Orange, Alexa Fluor® 405, and Pacific Blue[LifeTech] are clone S3.5 (Life Technologies, Carlsbad, CA); Krome Orange is clone 13B8.2 (Beckman-Coulter Immunotech SAS, Marseille, France); V500-C, V550, AmCyan, FITC, and R-PE are clone SK3 (BD Biosciences, San Jose); V500, Pacific Blue[BD], V450, and BV421[BD] are clone RPA-T4 (BD Biosciences, San Jose); BV570 and BV421[BioLegend] are clone RPA-T4 (BioLegend, San Diego, CA).

Surface staining

Normal human blood was collected from healthy anonymous volunteers under an internal BD Biosciences donor program in EDTA (BD Vacutainer® K3-EDTA; Becton Dickinson and Company, Franklin Lakes, NJ). The blood samples were incubated at ambient temperature for 30 min with mouse anti-human CD4, CD8, or CD45 (clones SK3, SK1, or 2D1) conjugates at concentrations from 0.125 to 1 µg of antibody for 100 µl of blood. After incubation with the antibody conjugate, the blood was suspended in a 20-fold volume (2 ml) of BD FACS™ lysing solution and incubated for 10 min at ambient temperature. The samples were centrifuged at 300× g for 5 min, the supernatant was decanted, and the pellet was resuspended and washed with 2 ml of 0.5% BSA/PBS, pH 7.2 (PBSA). The supernatant was again decanted and the pellet was resuspended in 0.5 ml of PBSA for analysis on a BD LSRII Flow Cytometer below).

Single color controls (cells stained with CD3 V450, CD4 V500, CD4 AmCyan, CD4 V550, CD8 V550, CD45 AmCyan, CD45 V500, and CD8 Alexa Fluor 488) were used to set compensation in multicolor experiments. For single color experiments, spillovers were calculated instead. V450 was detected in the 450/50 nm channel on the violet laser octagon (BD LSRII 405 Volts). V500, AmCyan, or V550 was detected in the 525/50 nm or 585/42 nm channel (BD LSRII 360 volts). The compensation matrix used on the BD LSRII was V500-V450, AmCyan-V450, and V550-V450, all 12.2%; V450-V500 28.0%; V450-AmCyan 45.8%; V450-V550 7.9%.

For each sample, 10,000 events were collected on a BD LSRII fitted with a 488 nm Argon laser, Red HeNe 633 nm diode and violet diode 405 nm. Data was analyzed with either FACSDiva Software Version 6.1.3 or FCS Express Version 4 (in the form of list-mode data files version FCS 3.0; De Novo Software, Los Angeles, CA). Instrument setup was performed by optimizing the PMT voltages for each detector so that the measured standard deviation of unstained cells had less than a 10% contribution of electronic noise and the positively stained populations were within the linear range of the detector. The standard deviation and the maximum linearity of the detector were assessed by using cytometer setup and tracking (CS&T) fluorescent beads (BD Biosciences, San Jose, CA) with the CS&T software module available on all digital flow cytometers running BD FACSDiva software. Lymphocytes were gated using a forward light scatter (FSC) versus 90° orthogonal light scatter (SSC) dot plot and median fluorescence intensity (MFI) was measured on the gated population. Stain index (SI), a measure of resolution sensitivity (the ability to resolve a dim positive signal from background), was calculated for all specificities except CD45.

  • display math

Median fluorescence intensity (MFI) of positive and background populations was used for calculation of SI.

Median fluorescence intensity (MFI) was shown for CD45 due to the lack of an unstained background population (because CD45 is present on lymphocytes, monocytes, and granulocytes) and the calculation of SI requires a background population ([7]).

All figures and tables reflect results of single-sample determinations.

Use of V500 (CD3 V500-C) in multicolor panels (surface and intracellular staining)

For 8-color analysis aimed at determining the combined CD27/CD45RA phenotype of IFN-γ, IL-2, and/or TNF-α producing CD8+ T-cells, heparinized whole blood was stimulated for 5 h with Staphylococcal enterotoxin B (SEB) in the presence of the secretion inhibitor Brefeldin A (both from Sigma-Aldrich, St. Louis, MO). The samples were subsequently stained for 30 min at room temperature with CD8 V450, CD3 V500-C, CD27 PerCP-Cy5.5, CD45RA PE-Cy7, and CD4 APC-H7 (BD Biosciences, San Jose). After treatment with FACS lysing solution and FACS permeabilizing solution 2 (BD Biosciences, San Jose), the samples were stained with anti-TNFα FITC, anti-IL-2 PE, and anti-IFNγ APC (BD Biosciences, San Jose, CA) following a standard intracellular cytokine assay protocol ([8]).

For 7-color cell analysis designed to measure transient phosphorylation of intracellular signaling proteins, whole blood was stained with CD27 FITC, CD45 RA PE-Cy7, CD4 APC-H7, CD8 V450, and CD3 V500-C and simultaneously activated with SEB (20 min, 37°C). The samples were then treated with BD Phosflow™ Lyse/Fix Buffer and Phosflow Perm Buffer IV. Intracellular staining with anti-phospho-p38 MAPK Alexa 647 and anti-phospho-ERK-1/2 PE (BD Biosciences, San Jose) was performed following an intracellular phospho-protein staining protocol ([9]).

For both panels, samples were analyzed on a FACSCanto II flow cytometer.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Physical Properties

Spectral characteristics, quantum yield, molar extinction coefficient, the influence of pH on fluorescence, and labeling efficiency all contribute to the success of a dye as a label in flow cytometry.

Absorption

The absorption characteristics of the dyes investigated are consistent with efficient excitation by a 405 nm violet laser line (Fig. 2A and Table 1). Specifically, both V500 and AmCyan have low absorption (10% or less of their maxima) at 488 nm (blue laser line), whereas FITC has very strong absorption (88% of maximum). Overlap in the absorption spectra of FITC and AmCyan in the wavelength region where FITC is excited on typical flow cytometers (488 nm) leads to the necessity of applying significant (cross-laser) compensation to the FITC detector when these two fluorophores are used simultaneously. Because the absorption spectrum of V500 is blue-shifted relative to that of AmCyan, its absorbance is only 25% of the absorption of AmCyan at 488 nm. When V500 is used instead of AmCyan in FCM, the result is much less excitation from the blue laser, and hence, significantly less spillover into FITC detector. FCM experiments confirmed that V500 reagents had much less spillover into FITC compared with AmCyan reagents (discussed in greater detail in Flow Cytometric Evaluation section).

image

Figure 2. A: Normalized absorption spectra of violet-excitable dyes. Note that the violet laser line (405 nm; dashed purple line) strikes the absorption peaks of these dyes (except for AmCyan) at a height of 95% or more of maximum. Both V500 and AmCyan have low-efficiency blue-laser (blue dashed line) excitability, but V500 is lower than AmCyan. B: Normalized emission spectra of violet-excitable dyes. Note the relative positions of the emission spectra with regard to the violet and blue laser lines (dashed lines), and that bandpass filters must avoid these lines so as not to admit stray light to the detectors which they govern.

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Table 1. Spectral characteristics, quantum yields, and ph dependence of violet-excitable dyes
DyeExcitation maximum (nm)Emission maximum (nm)Stokes shift (nm)Molar extinction coefficient (ε, M−1 cm−1)Quantum yield (QY)Brightnessa (M−1 cm−1)pKab or compatible pH range
  1. a

    Brightness=ε × QY.

  2. b

    pKa is the pH value at which 50% of the maximum fluorescence is attained; it is assumed that maximum fluorescence will be attained above this value and be stable within the biological pH range.

  3. c

    Sirigen Inc.

  4. d

    Not determined; manufacturer cannot provide this information but some characterization has been published (10).

  5. e

    Ref. [4].

  6. f

    Figure 4.

  7. g

    Ref. [11].

  8. h

    Ref. [12].

  9. i

    Data not shown in text

  10. j

    Ref. [13].

  11. k

    Not available; proprietary information that manufacturer will not provide, but a detailed characterization has been published (14).

  12. l

    Ref. [15].

  13. m

    Ref. [16].

BV421407421142,500,000c0.65c1,625,000cND
Alexa Fluor 4054004242434,0000.5418,900NDd
V4504054504538,5000.9837,730pKa = 6.4e, f
Pacific Blue4164513546,0000.75g34,500pKa = 3.7g
AmCyan45848931158,000 (tetramer)0.42h66,360About 8% fluorescence loss at pH 10 versus pH 4.5i
V5004145008628,5000.8624,510About 13% fluorescence loss at pH 6.5 versus pH 10f
Fluorescein4945212770,0000.9365,100pKa = 6.4j
Krome Orange3985283017,665NAkNAkNAk
V55040553513020,5000.6813,940Stable fluorescence from pH 5.0 to pH 9.5f
Pacific Orange40455314925,000NDdNDdAbout 9% fluorescence loss at pH 11 versus pH 4.0i
BV570407570632,300,000c0.080c184,000cND
R-PE56657591,992,0000.82l1,633,440Stable fluorescence from pH 3.5 to pH 10m

Emission

The emission spectra of the dyes along with the relative positions with regard to the violet and blue laser lines (dashed lines; Fig. 2B) demonstrate certain restrictions in choosing bandpass filters and attempting to use AmCyan or V500 together with V550. In particular, filters must avoid the laser lines so as not to admit stray light to the detectors that they govern. Therefore, detectors intended for V500 and AmCyan emission collection must have nonideal filters, which pass only light of higher wavelength than about 500 nm, rejecting a significant portion of their emission peaks.

Quantum Yield

Quantum Yield (QY) is one of the most important properties used to characterize a new fluorophore; it measures the efficiency of the fluorescence process and is defined as the ratio of the number of photons emitted to the number of photons absorbed. The maximum fluorescence QY is 1.0 (100%) when every photon absorbed results in a photon emitted. The QYs of V450, V500, and V550 were determined to be 0.98, 0.86, and 0.68, respectively (Table 1) which makes them exceptionally efficient fluorophores. In our previous study ([4]), we showed that V450, a monochlorinated hydroxycoumarin, made somewhat brighter antibody conjugates than the fluorinated analog Pacific Blue, but we incorrectly stated that the QY for V450 was equal to that of Pacific Blue. In the current study, we display the QYs of Pacific Blue (0.75) and V450 (0.98) in Table 1, and observe that the increased brightness of the V450 antibody conjugates may be related to V450′s higher QY.

Molar Extinction Coefficient

The molar extinction coefficient (ε), which represents the ability of a fluorophore to absorb light of a specified wavelength, and QY are equally important indicators of dye functionality because brightness is the product of ε and QY. Molar extinction coefficients and QYs of V450, V500, and V550 are compared with other violet-excitable dyes in Table 1. Although violet-excitable organic dyes have significantly lower molar extinction coefficients than R-phycoerythrin (R-PE), they are still capable of producing bright reagents. As we have shown, it is possible to attach as many as 50 molecules of these dyes to selected antibody molecules (e.g., CD8; see Fig. 3A) compared with only 1 or 2 R-PE molecules ([17]), the lower molar extinction coefficients and QYs can be offset.

image

Figure 3. A: Stain Index or MFI from cell staining of CD3, CD4, CD8, and CD45 V500 at different D/P ratios. B: Stain Index from cell staining of CD4 V550 and Pacific Orange. C: MFI from cell staining of CD45 V550 and Pacific Orange.

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pH Range

FCM reagents must perform optimally in a pH range of 6.5 to 7.5, analogous to pH ranges observed under physiological conditions. Many organic dyes lack this property, and even those that have been rendered water-soluble may not be compatible with a neutral pH ([18]). Fortunately, V450, V500, and V550 meet this criterion for compatibility with biological systems. V450 and V500 attain greater than 85% of their maximum fluorescence at pH 6.5, and V550 shows a constant level of fluorescence output over a pH range of 5 to 9.5 (Fig. 4).

image

Figure 4. pH dependence of V450, V500, and V550 fluorescence. All three dyes had characteristics compatible with use in cell staining; namely fluorescence output close to their maxima at pH 7 and above.

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Labeling Efficiency

The maximum number of dye molecules that can be attached to a given antibody clone can make the difference between a successful or unsuccessful dye conjugate, and a low labeling efficiency can be responsible for the dimness of certain dyes on certain antibodies. Labeling efficiency was a major consideration in the evaluation of our violet dyes, and comparisons have either been reported between V450 and Pacific Blue ([4]) or presented between V550 and Pacific Orange (Fig. 3), demonstrating that, for certain antibodies, higher numbers of V450 or V550 dye molecules can be attached compared with the number of Pacific Blue or Pacific Orange molecules that can be attached.

Flow Cytometric Evaluation

Although characterization of the physical properties of a new dye or reporter can predict functionality, FCM testing is essential to optimize labeling conditions and to confirm its actual functionality. For FCM users preparing their own reagents, dye loading or labeling levels are of interest. For users of the reagents who will start with purchased or fully optimized reagents, performance oriented results are more important. These results include brightness, spillover and compensation, and the ability to use reagents together in panels, either with other reagents excited by a common laser or those excited with different lasers.

A discussion of the optimization of V450 labeling of three antibodies has been previously reported ([4]). V500-antibody conjugates from some of the same common cell-surface markers used for V450, i.e., CD3 (SK7), CD4 (SK3), CD8 (SK1), and CD45 (2D1), were made and subjected to similar testing in this study. The performance characteristics revealed the following: CD3 and CD45 displayed a peak of D/P ratios with optimal performance at around 5 and 9, respectively. Optimal performance for CD4 and CD8 could be produced at D/P ratios from 24 to 37 for CD4 and from 28 to 50 for CD8 (Fig. 3A). On the basis of these observations, optimal D/P ranges were identified as 3 to 7 for CD3, 22 to 30 for CD4, 26 to 34 for CD8, and 6 to 10 for CD45.

SI results from cell staining of CD4 V550 and Pacific Orange (Fig. 3B) reveal that substantially more V550 dye molecules can be attached to CD4 versus Pacific Orange (about 35 vs. 15). As the SI continues to rise as more dye molecules are attached, V550 makes a better performing CD4 reagent than does Pacific Orange. MFI results from cell staining of CD45 V550 and Pacific Orange show that, although equal numbers of dye molecules of both V550 and Pacific Orange can be attached to CD45, the performance falls off as the number of V550s increases, in contrast to Pacific Orange, making Pacific Orange a better choice for a CD45 reagent than V550.

V500- and AmCyan1-antibody conjugates were compared for spillover into both FITC and V450 channels. The spillovers from V500 conjugates into both FITC and V450 channels (blue laser 525/50 and violet laser 450/50 detectors, respectively) were all consistently lower than AmCyan conjugates. For instance, when CD4 V500 was substituted for CD4 AmCyan, spillover into the FITC channel fell from 90.5% to 8.6% and spillover into the V450 channel fell from 4.5% to 3.1%. These results demonstrate that V500 has better compatibility with V450 and FITC for multicolor applications than does AmCyan.

CD4 conjugates of violet-excitable dyes from various sources were compared in lyse-wash staining of whole blood (Fig. 5). FITC and PE were also included for reference. All of the conventional violet-excitable fluorophores, comprising either organic dye or fluorescent protein, were dimmer than either FITC (with 488 nm excitation), PE (with 561 nm excitation), or fluorescent polymers and polymer-dye tandems such as BV421 and BV570 (BD Biosciences or BioLegend). When excited with a violet laser, these polymer-based conjugates were nearly as bright as PE when PE was optimally excited with a 561 nm yellow-green laser. SIs for Krome Orange, Pacific Orange, and V550 were not affected by the choice of 525/50 or 582/15 nm bandpass filtered detection, so the results from 525/50 were plotted.

image

Figure 5. The CD4 conjugates across dye types, lasers, and manufacturers ranked by Stain Index. Most violet-excitable dyes, including V450, V500, and V550, exhibit poorer performance than FITC or PE. The brightest violet-excitable reagents available at this time are BrilliantViolet™ (Sirigen, San Diego, CA) based (e.g. BV421; BioLegend, San Diego, CA; BD Biosciences, San Jose, CA), and are of similar brightness to PE. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The BD Horizon violet-excitable dye-CD4 conjugates were midrange in brightness compared with other CD4-violet-excitable dye conjugates except for the much brighter conjugates made with BrilliantViolet™ dyes (Fig. 5).

The violet-excitable dyes described in this report can be classified into three groups based on their emission maxima (Table 1). BV421, Alexa Fluor 405, V450, and Pacific Blue have the lowest emission maxima (EM, 421–451 nm), AmCyan, V500, and Krome Orange have slightly higher EM (489–528 nm) and V550, Pacific Orange and BV570 have the highest EM (535–575 nm). V450, V500, and V550 place into the first, second and third groups, respectively, midway between brighter and dimmer alternative dyes.

Examples of a 3-color combination (CD3 V450/CD4 V500/CD8 V550; Fig. 6) and three different 2-color staining combinations (CD3 V450/CD4 V500, CD3 V450/CD4 AmCyan, and CD3 V450/CD4 V550; Fig. 7) are shown. Both V500 and V550 required much less compensation than did AmCyan. A 582/15 nm bandpass filter was used for detection of V550.

image

Figure 6. V450, V500, and V550 used in combination to identify CD4 and CD8-positive T-cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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image

Figure 7. V450 used in combination with either V500, V550, or AmCyan to identify CD4-positive T-cells.

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Two-color results comparing V500 and AmCyan (Fig. 8) demonstrated that because V500 required much less compensation out of the blue-laser 530/30 detector than did AmCyan, the sensitivity of the CD8 Alexa Fluor 488 reagent was greatly enhanced, allowing the CD8+ (bright) population to be resolved from the negative population.

image

Figure 8. Improved resolution of CD8 Alexa Fluor® 488 when using CD45 V500 rather than AmCyan. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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In addition to direct labeling of antibodies with V500, we developed V500-C conjugation. The V500-C chemistry was devised because of the inability to set compensation accurately using the BD CompBeads set ([5]) (data not shown). This conjugation chemistry (labeling a small protein with V500 and SMCC and then conjugating that modified protein to antibodies) mitigated the problem for V500. For the studies in this report, CD3 V500-C was titrated on live cells and on cells that had been fixed and permeabilized and performed optimally at a concentration of 0.125 µg and 0.016 µg per 100 µl of blood, respectively. An 8-color cytokine assay and a 7-color signaling protein transient phosphorylation analysis were then run using the CD3 V500-C to gate on T cells. In both cases, the CD3+ population was clearly identified, allowing facile gating and additional subdivision of the gated population into CD4+ and CD8+ subsets, with further gating of CD8+(bright) T-cells. The gated bright CD8+ population was then analyzed, determining the combined CD27/CD45RA phenotype of IFN-γ, IL-2, anD/or TNF-α producing cells (Fig. 9) or phospho-p38 MAPK and phospho-ERK-1/2 producing cells (Fig. 10).

image

Figure 9. Whole blood stimulated with SEB in the presence of Brefeldin A. CD3 V500-C is used to gate T-cells, which are further subsetted.

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image

Figure 10. Whole blood simultaneously activated with SEB and stained with surface markers followed by permeabilization and intracellular staining of phosphor-proteins.CD3 V500-C is used to gate T-cells, which are further subsetted.

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These results demonstrate that V500 reagents can be used in multicolor assays where cells undergo complex treatments involving multiple steps and reagents (e.g., SEB or Brefeldin A stimulation, surface and intracellular staining, lysis, fixation, and permeabilization).

Other Findings

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

The initial yields of V500-antibody conjugates made from hydrophobic clones (those with a history of poor solubility upon conjugation with dyes) were unacceptably low (15% or less of the starting antibody). Studies were performed evaluating the effects of pH, salt concentration, and polarity-modifying agents, such as glycerol, DMSO, and ß- hydroxypropylcyclodextrin on maintaining solubility during labeling, purification, and storage at 2 to 8°C. Solubility was assessed visually (for cloudiness and spontaneous formation of a pellet), as well as quantitatively, by centrifugation and spectrophotometric analysis of the supernatant. From the various combinations evaluated, adding glycerol and raising the pH were judged most effective at preserving solubility of purified concentrated conjugate in the cold. Yields of conjugate, without regard to clone, were typically 50% or higher with 20 % (v/v) glycerol in a pH 8.3 storage buffer.

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

V450, V500, V550, and AmCyan are all suitable as labels for violet-excitable FCM reagents. These dyes are easily conjugated to antibodies, have absorption characteristics consistent with violet-laser excitation, and are fluorescent at 450–535 nm. CD4-antibody conjugates derived from these three dyes were comparable in brightness to other violet-excitable conjugates in the market with the exception of the very bright conjugates made with BrilliantViolet™ that are as bright as PE conjugates. V500 and V550 required much less compensation than did AmCyan. Spillover from V500-antibody conjugates into the blue laser (525/50) and the violet laser (450/50) detectors was consistently lower than spillover from AmCyan conjugates; an average of 74% lower into the blue laser 530/30 detector and 1% lower into the violet laser 440/40 nm detector, showing that V500 was more compatible with FITC and V450 than was AmCyan for multicolor applications.

V500 has unique spectral properties; specifically a 500 nm emission maximum with an 80 nm emission peak half-height, making it the only fluorophore of its kind with no commercially available analog. V500-antibody conjugates are clearly well suited for use in multicolor panels and are compatible with protocols employing FACS lysing solution as well as fixing and permeabilizing reagents necessary for intracellular staining. Thus, V500 makes an ideal second color for violet excitation in FCM, either directly conjugated to antibody or in conjunction with a protein carrier, whereby accurate compensation of cells using BD CompBeads for instrument setup can be achieved.

In addition to their use in FCM, the dyes described in this report could be used in conjunction with a panel of QDots on a less expensive single-laser flow platform (19,20). One possible design is a panel consisting of one of the 2-color combinations discussed above (Fig. 7) (composed of fluorophores with emission maxima from 450 to 550 nm covering the blue-orange spectral region) and three QDots with emission maxima at 655, 707, and 800 nm (covering the red-infra red region). This 5-color panel would require minimal compensation and combines the low nonspecific binding and solubility of small organic dyes or fluorescent proteins with the brightness and long Stokes shifts of QDots for use on a portable, inexpensive platform that would enable multi-parameter analysis of biological samples.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

The authors thank Dr. Nancy Gadol for review and editing of the manuscript, and Dr. Maria C. Jaimes for her patient and thorough introduction to “Godot,” the lab's LSRII.

Literature Cited

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  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Other Findings
  6. Conclusions
  7. Acknowledgments
  8. Literature Cited
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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