Molecular Probes

Section 15.4 — Assays for Cell Enumeration, Cell Proliferation and Cell Cycle

Cell proliferation and the characterization of agents that either promote or retard cell proliferation are extremely important areas of cell biology and drug-discovery research. Molecular Probes offers both traditional reagents for assessing cell proliferation and cell cycle — in particular the Hoechst nucleic acid stains and probes for 5-bromo-2'-deoxyuridine (BrdU) incorporation during cell division — as well as some exceptional tools that we have developed, including our CyQUANT GR, TOTO-1, SYTOX Green and SYTO dyes. For simply detecting the presence of cells or enumerating them, fluorescent stains that identify cells by their characteristic morphology or light-scattering properties may be sufficient. For example, some of our SYTO dyes have been employed to detect microorganisms that could be used as biological weapons. The sensitivity of some of our fluorescent dyes even permits the detection and quantitation of viruses under certain circumstances.

Cell Enumeration and Cell Proliferation Assays for Animal Cells

Reagents for counting cells and quantitating cell proliferation are valuable research and diagnostic tools. Currently there is no fluorescent reagent that can be specifically incorporated into cells during cell division and directly detected on a single cell basis. Consequently, most cell proliferation assays estimate the number of cells either by incorporating ³H-thymidine or 5-bromo-2'-deoxyuridine (BrdU, a thymidine analog; B23151) into cells during proliferation or by measuring total nucleic acid or protein content of lysed cells. Several of our nucleic acid stains (Section 8.1) and nucleotides (Section 8.1) have proven useful in BrdU-labeling protocols. Our CyQUANT Cell Proliferation Assay Kits (C7026, C35007, C35006) use the CyQUANT GR or CyQUANT NF reagent to measure changes in nucleic acid content that occur during cell proliferation.

Proliferation Assays Using Bromodeoxyuridine Incorporation

Incorporation of 5-bromo-2'-deoxyuridine (BrdU, B23151) into newly synthesized DNA permits indirect detection of rapidly proliferating cells with fluorescently labeled anti-BrdU antibodies or certain nucleic acid stains, thereby facilitating the identification of cells that have progressed through the S-phase of the cell cycle during the BrdU labeling period. In conjunction with Phoenix Flow Systems, Molecular Probes offers fluorescent conjugates of the mouse monoclonal anti-BrdU antibody PRB-1 (Anti-bromodeoxyuridine Antibodies) labeled with our brightest and most photostable dyes — the Alexa Fluor 488 (A21303), Alexa Fluor 532 (A21307), Alexa Fluor 546 (A21308), Alexa Fluor 594 (A21304), Alexa Fluor 647 (A21305), Alexa Fluor 660 (A21306) and Alexa Fluor 680 (A31859) dyes. This anti-BrdU antibody is also available as a biotin-XX conjugate (A21301MP), as well as unlabeled (A21300). The unlabeled mouse anti-BrdU can be detected with our anti-mouse secondary antibodies (Table 7.1) by either flow cytometry (Figure 15.59) or imaging (). In addition to its use for detecting BrdU-labeled DNA, monoclonal PRB-1 recognizes 5-bromouridine (BrU) incorporated into RNA and the same anti-BrdU conjugates can be used for the specific staining of RNA in cells that has incorporated BrU. It should be possible to amplify the detection of very low degrees of BrdU incorporation by using the biotin-XX conjugate of anti-BrdU (A21301MP) in conjunction with one of our streptavidin-based Tyramide Signal Amplification (TSA) Kits, which are described in Section 6.2. The unlabeled anti-BrdU antibody may also be used (A21300) with any of the Zenon Mouse IgG₁ Labeling Kits (Section 7.3, Table 7.14, ).

Because fluorescence of the Hoechst 33258 (H1398; H3569; FluoroPure Grade, H21491) and Hoechst 33342 (H1399; H3570; FluoroPure Grade, H21492) dyes bound to DNA is quenched at sites where BrdU is incorporated, Hoechst dye fluorescence can also be used to detect BrdU incorporation in single cells. This technique has been employed to quantitate the noncycling cell fraction, as well as the fraction of cells that are in G₁ and G₂ of two subsequent cycles. The addition of ethidium bromide (E1305, E3565; Section 8.1) as a counterstain that is insensitive to BrdU incorporation allows the resolution of G₁, S and G₂ compartments of up to three consecutive cell cycles.

Unlike the fluorescence of Hoechst dyes, the fluorescence of TO-PRO-3 (T3605) and LDS 751 (L7595) is considerably enhanced by the presence of bromodeoxyuridine in DNA. In conjunction with propidium iodide (P1304MP, P3566, P21493; Section 8.1), these nucleic acid stains have been used to discriminate BrdU-labeled cells from nonproliferating cells by flow cytometry and with an imaging system for automated cell proliferation.

Proliferation Assays Using ChromaTide Nucleotides

In the strand break induction by photolysis (SBIP) technique, proliferating cells that have incorporated BrdU into newly synthesized DNA are subjected to Hoechst 33258 staining, followed by UV photolysis to induce DNA strand breaks (Figure 15.62). Once the cells are fixed, strand breaks can be detected in situ using mammalian terminal deoxynucleotidyl transferase (TdT), which covalently adds labeled nucleotides to the 3'-hydroxyl ends of these DNA fragments. Break sites have traditionally been labeled with biotinylated or haptenylated dUTP conjugates (Section 8.2) in conjunction with antibodies to the hapten (Section 7.4) or conjugates of streptavidin (Section 7.6). However, a single-step procedure has been described that uses our ChromaTide BODIPY FL-14-dUTP (C7614, ) as a TdT substrate for directly detecting DNA strand breaks both in BrdU-labeled cells following SBIP and in apoptotic cells (Section 15.5; , ). The single-step BODIPY FL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides. With direct detection procedures, no secondary detection reagents are required; fewer protocol steps translate into less chance for error and more immediate results. Moreover, the yield of cells with direct detection procedures is reported to be about three times greater than that of multistep procedures employing biotin- or digoxigenin-conjugated dUTP. Although both BODIPY FL dye– and fluorescein-labeled nucleotides can be detected with fluorescence microscopy or flow cytometry, the BODIPY FL dye–labeled nucleotides provide ~40% stronger signal than fluorescein-labeled nucleotides when assaying strand breaks in apoptotic versus nonapoptotic cells. In addition, fading of the fluorescence of the incorporated BODIPY FL dUTP is less than that of the corresponding fluorescein dUTP analog. Unlike traditional proliferation assays based on BrdU incorporation, no DNA heat- or acid-denaturation steps are required with SBIP in order to visualize the labeled strand breaks, allowing simultaneous detection of the morphology of nuclear proteins and other cellular constituents by immunocytochemical analysis. The narrow emission spectrum of the BODIPY FL dye–labeled nucleotides is especially useful for multicolor labeling experiments.

An elegant technique permits tracking of labeled chromosomes through mitosis by metabolic incorporation of microinjected fluorescent nucleotides, including our fluorescein-12-dUTP, Oregon Green 488-5-dUTP and BODIPY TR-14-dUTP (C7604, C7630, C7618; Section 8.2), by endogenous cellular enzymes into DNA. The procedure does not interfere with subsequent progress through the cell cycle, and fluorescent strands of DNA can be followed as they assemble into chromosomes and segregate into daughters and granddaughters. Presumably, injection of 5'-bromo-2'-deoxyuridine triphosphate (BrdUTP, B21550), followed by detection of the incorporated BrdU with one of our Alexa Fluor conjugates of anti-BrdU would also be suitable for studying mitosis. The corresponding ribonucleotide (BrUTP, B21551) that has been microinjected into cells is incorporated into RNA of a nucleolar compartment, a process that should also be detectable with fluorescent anti-BrdU conjugates.

ABSOLUTE-S SBIP Cell Proliferation Assay Kit

Many conventional BrdU-based protocols for assaying cell proliferation require DNA denaturation in order for the BrdU epitope to become accessible to the anti-BrdU antibody. The DNA denaturation is typically accomplished by heat (>90°C) or strong acid (2–4 M HCl). Such harsh treatments often make it difficult to perform multiparameter analysis because other cellular structures and antigens are not well preserved during these treatments.

In conjunction with Phoenix Flow Systems, Molecular Probes offers the ABSOLUTE-S SBIP Cell Proliferation Assay Kit (A23150), which utilizes the strand break induction by photolysis (SBIP) methodology described above and does not require DNA denaturation at any step. In the ABSOLUTE-S assay, cells are first incubated in the presence of the BrdU photolyte, which is incorporated into cellular DNA during replication. Next, the photolyte enhancer is added to the cell culture to sensitize the BrdU-labeled DNA to photolysis. Once this treatment is complete, the cells are irradiated by UV light to induce DNA strand breaks at the sites where BrdU is incorporated. Additional BrdU is then added at the break sites using the TUNEL (terminal deoxynucleotidyltransferase dUTP nick-end labeling) technique. Finally, BrdU is detected using an Alexa Fluor 488 dye–labeled anti-BrdU monoclonal antibody (). This kit also provides propidium iodide for determining total cellular DNA content, as well as fixed control cells for assessing assay performance.

The ABSOLUTE-S Kit includes complete protocols for use in flow cytometry applications, though it may also be adapted for use with fluorescence microscopy. Each kit contains:

• BrdU photolyte, for labeling the cellular DNA during replication
• Photolyte enhancer, for sensitizing the BrdU-labeled DNA to UV photolysis
• Terminal deoxynucleotidyl transferase (TdT), for catalyzing the addition of BrdUTP at the break sites
• 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP)
• Alexa Fluor 488 dye–labeled anti-BrdU mouse monoclonal antibody PRB-1, for detecting BrdU labels
• Propidium iodide/RNase staining buffer, for quantitating total cellular DNA
• Reaction, wash and rinse buffers
• Reaction control cells (a fixed human lymphoma cell line)
• Photolysis control cells (a fixed human lymphoma cell line)
• A detailed protocol (ABSOLUTE-S SBIP Cell Proliferation Assay Kit)

Sufficient reagents are provided for approximately 60 assays of 1 mL samples, each containing 1–2 × 10⁶ cells/mL.

Proliferation Assay Using the Succinimidyl Ester of Carboxyfluorescein Diacetate and Related Probes

The succinimidyl ester of carboxyfluorescein diacetate (5(6)-CFDA, SE or CFSE, C1157) is currently the most widely used probe for generation analysis of cells, although our succinimidyl ester of Oregon Green 488 carboxylic acid diacetate (O34550, C34555; see below) offers several important advantages over this fluorescein derivative. CFDA SE spontaneously and irreversibly couples to both intracellular and cell-surface proteins by reaction with lysine side chains and other available amine groups. When cells divide, CFDA SE labeling is distributed equally between the daughter cells, which are therefore half as fluorescent as the parents. As a result, each successive generation in a population of proliferating cells is marked by a halving of cellular fluorescence intensity (excitation/emission maxima ~495/525 nm) that is readily detected by a flow cytometer (Figure 15.66), fluorescence microscope or fluorescence microplate reader. CFDA SE is available as a single vial containing 25 mg (C1157). CFDA SE is also available conveniently packaged for cell tracing applications in our Vybrant CFDA SE Cell Tracer Kit (V12883, Figure 15.66) and for cell proliferation studies in our CellTrace Cell Proliferation Kit (C34554, Figure 15.67). The fluorescent CFDA SE product has excitation/emission maxima of ~492/517 nm and can be detected using a fluorescence microscope, flow cytometer or fluorescence microplate reader. Each kit includes 10 single-use vials of CFDA SE (500 µg each in Kit V12883, 50 µg each in Kit C34554), as well as high-quality anhydrous DMSO and a complete protocol (Vybrant(R) CFDA SE Cell Tracer Kit, CellTrace CFSE Cell Proliferation Kit).

CFDA SE produces more homogenous cellular labeling and, consequently, much better intergenerational resolution than other cell-tracking dyes, such as the membrane marker PKH26. Using flow cytometric analysis of CFDA SE labeling, researchers can reliably resolve 8 to 10 successive generations of lymphocytes. In transplanted cells the signal of CFDA SE can be traced in vivo for weeks. The potential of this important technique is discussed in a collection of nine articles and reviews appearing in the December 1999 issue of Immunology and Cell Biology. The feasibility of using cell-permeant fluorescent tracers to follow cell division of natural killer (NK) cells, B cells, T cells, thymocytes, lymphocytes, fibroblasts and hematopoietic cells has been demonstrated with CFDA SE. For instance, researchers have used CFDA SE labeling to show that transplantable hematopoietic cells proliferate in vitro in response to stimulation by a growth factor cocktail. These studies helped provide direct evidence that the hematopoietic potential of cultured stem cells is limited by homing activity and not by proliferative capacity. Because the first division results in the largest change in fluorescence intensity, this method is particularly useful for detecting subsets of cells within a population that are resistant to cell division. The method is not limited to mammalian cells; it has also been applied to determine the number of cell divisions in stained Lactobacillus plantarum.

Like CFDA SE, the succinimidyl ester of Oregon Green 488 carboxylic acid diacetate (carboxy-DFFDA SE) should be a useful tool for following proliferating cells. This Oregon Green 488 probe passively diffuses into cells, where it is colorless and nonfluorescent until its acetate groups are removed by intracellular esterases to yield a highly green-fluorescent, amine-reactive dye. Upon reaction with intracellular amines, the probe forms Oregon Green 488 conjugates that are well-retained by cells. Unlike fluorescein derivatives, however, Oregon Green 488 derivatives exhibit bright green fluorescence that is not pH dependent at typical cellular pH values. Moreover, Oregon Green 488 probes are usually brighter and more photostable than fluorescein probes. We offer carboxy-DFFDA SE in a 1 mg unit size (O34550) and specially packaged in a set of 20 vials, each containing 50 µg (CellTrace Oregon Green 488 carboxylic acid diacetate succinimidyl ester, C34555).

The intracellular conjugates of 5-(and 6-)carboxyeosin diacetate succinimidyl ester (C22803) have absorption and emission spectra at longer wavelengths than CFDA SE, which may make this probe useful in combination with CFDA SE for studies of proliferation of mixed-cell populations. Eosin conjugates are more effective singlet-oxygen generators than are simple fluorescein derivatives, potentially resulting in their utility for photoablation of cells.

The succinimidyl ester of SNARF-1 carboxylic acid, acetate (S22801, ) is also designed to serve as a cell tracer and indicator of cell division. However, unlike the green-fluorescent CFDA SE–labeled cells, cells labeled with the succinimidyl ester of SNARF-1 carboxylic acid, acetate exhibit red fluorescence when excited near 488 nm. Although the fluorescence intensity of this SNARF derivative in cells may be weaker than that of cells labeled with CFDA SE, its red fluorescence is easily distinguished from the green fluorescence of CFDA SE–labeled cells. The SNARF dyes have been predominantly used as indicators of intracellular pH (Chapter 20).

Vybrant DiI Cell-Labeling Solution

Analysis by mass spectrometry and HPLC indicates that the dye we use in our Vybrant DiI cell-labeling solution (V22885) is identical to the dye called PKH 26. DiI (PKH 26) is a red-fluorescent lipophilic tracer that, in addition to being extensively used for cell tracing (Section 14.4), has been utilized for generational analysis of cells undergoing division. Unlike the PKH 26 dye, which requires a special cell-labeling medium and low ionic strength for successful cell loading, our Vybrant DiI cell-labeling solution is simply added to cells in normal growth medium (Vybrant(R) Cell-Labeling Solutions). Dividing cells distribute the lipophilic tracer approximately equally between daughter cells. It is usually possibly to follow at least three or four generations of cells by flow cytometry, although asynchronous division times can quickly complicate the measurements. The dyes in our Vybrant DiO, Vybrant DiD and Vybrant CM-DiI Labeling solutions (V22886, V22887, V22888) may have similar utility for tracing cells through cell division. CM-DiI contains a thiol-reactive chloromethyl that allows the dye to covalently bind to cellular thiols. Thus, unlike other membrane stains, this label is well retained in some cells throughout fixation and permeabilization steps; see Section 14.4 for more information.

CyQUANT Cell Proliferation Assay Kit

Because cellular DNA content is highly regulated, it is closely proportional to cell number. Therefore, changes in nucleic acid content can serve as a sensitive indicator of overall cell proliferation, as well as of cytotoxic events or pathological abnormalities that affect cell proliferation. Our CyQUANT Cell Proliferation Assay Kit (C7026) provides an excellent method both for enumerating cells in a population and for measuring their proliferative activity. This assay is an important development for the rapid and quantitative screening of agents that affect cell proliferation. The CyQUANT assay is based on the use of our Patented green-fluorescent CyQUANT GR dye, which exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The assay protocol is simple: the culture medium is removed (nonadherent cells require brief centrifugation); the cells are frozen, thawed and lysed by addition of the CyQUANT cell buffer containing detergent and the CyQUANT GR dye; and fluorescence is then measured directly in a fluorometer or fluorescence microplate reader (Figure 15.68). No washing steps, growth medium changes or long incubations are required. The CyQUANT cell proliferation assay has a number of significant advantages over other proliferation assays:

• Sensitivity and linearity. The CyQUANT assay is linear from 50 or fewer cells to at least 50,000 cells in 200 µL volumes (Figure 15.69); increasing the dye concentration extends the linear range to at least 250,000 cells. Methods that employ Hoechst 33258 (H1398, H3569; FluoroPure Grade, H21491) or Hoechst 33342 (H1399; H3570; FluoroPure Grade, H21492) to measure cell number and proliferation are much less sensitive — detection limits of 500 cells for Hoechst 33258 or 2500 cells for Hoechst 33342 — and have much smaller effective ranges.
• No radioactivity. Unlike assays that measure ³H-thymidine incorporation, the CyQUANT assay does not require radioisotopes and thus does not have the hazards or the expense associated with use, storage and disposal of radioisotopes.
• Quick and easy protocol. The CyQUANT assay is a single-step procedure that requires no lengthy incubation steps and can be completed within an hour (Figure 15.68).
• Specificity and reliability. The assay is specific for total nucleic acids, with essentially no interference from other cell components. No wash steps are required because cellular growth media do not significantly interfere with CyQUANT GR fluorescence. The CyQUANT assay is reliable for cell quantitation, even without treatment to eliminate cellular RNA. However, addition of RNase or DNase permits the easy quantitation of DNA or RNA, respectively, in the sample.
• Convenience. Unlike assays that use tetrazolium salts, ³H-thymidine, BrdU, neutral red or methylene blue, the CyQUANT procedure is not dependent on cellular metabolism. Thus, cells can be frozen and stored prior to assaying, with no reduction in signal, or they can be assayed immediately after collection. Time-course assays are simplified because data obtained from stored samples taken at widely different time intervals can be assayed together with a single standard curve determination.

We have found the CyQUANT Cell Proliferation Assay Kit to be useful for assaying widely disparate cell types, including:

• Human neonatal fibroblasts, keratinocytes, melanocytes, umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (DMVEC)
• Murine fibroblasts (NIH 3T3 and CRE BAG 2 cells) and myeloma (P3X63A68) cells
• Madin–Darby canine kidney (MDCK) cells
• Chinook salmon embryo (CHSE) cells
• Rat basophilic leukemia (RBL) and glioma (C6) cells

In addition to quantitating proliferation, the CyQUANT GR reagent may supplant ⁵¹Cr-release studies for monitoring T-cell cytolysis and other cytolytic events. Furthermore, determination of total cell number using the CyQUANT GR reagent is potentially useful for quantitating cell adhesion (see "Cell Adhesion" in Section 15.6) and for determining the total number of cells in a tissue. Each CyQUANT Cell Proliferation Assay Kit (C7026) includes:

• CyQUANT GR reagent
• Cell-lysis buffer
• DNA standard for calibration
• Detailed protocol (CyQUANT(R) Cell Proliferation Assay Kit)

The kit supplies sufficient materials for performing 1000 assays based on a 200 µL sample volume or a proportionately lower number of assays with a larger sample volume. The CyQUANT cell-lysis buffer (a 20X concentrate, C7027) is also available separately and has been formulated to produce efficient lysis, to protect nucleic acids from nuclease activity and to dissociate proteins that may interfere with dye binding to nucleic acids. It may prove generally useful in the development of other assays that require cell lysis.

CyQUANT NF Cell Proliferation Assay Kit

The CyQUANT NF Cell Proliferation Assay Kit provides a fast and sensitive method for counting cells in a population and measuring proliferation in microplate format. This assay can be completed in one hour, with no washes, cell lysis, long incubations or radioactivity required, and it is not dependent on physiological activities that may exhibit cell number–independent variability. The CyQUANT NF assay eliminates the freeze-thaw cell lysis step of the original CyQUANT cell proliferation assay by using a cell-permeant DNA-binding dye in combination with a plasma membrane–permeabilization reagent. The CyQUANT NF assay protocol requires only aspiration of growth medium (for adherent cells), replacement with dye binding solution, incubation for 30–60 minutes and then measurement of fluorescence in a microplate reader. The CyQUANT NF assay has a linear detection range from at least 100 to 20,000 cells per well in most cell lines using a 96-well microplate format and a 100 µL assay volume.

The CyQUANT NF Cell Proliferation Assay Kit can be used with either a 96-well or 384-well microplate format and is available in two configurations: a 200-assay kit (C35007) and a 1000-assay kit (C35006) for high-throughput applications. Each kit contains:

• CyQUANT NF dye reagent
• Dye delivery reagent
• Concentrated Hank's balanced salt solution (HBSS)
• Detailed protocol (CyQUANT(R) NF Cell Proliferation Assay Kit)

Vybrant MTT Cell Proliferation Assay Kit

Molecular Probes' convenient Vybrant MTT Cell Proliferation Assay Kit (V13154) simplifies the task of counting cells with a microplate absorbance reader. The colorimetric MTT assay, developed by Mossman, is based on the conversion of the water-soluble MTT to an insoluble purple formazan. This formazan is then solubilized, and its concentration determined by optical density at 570 nm. The Vybrant MTT Cell Proliferation Assay Kit provides a sensitive assay with excellent linearity up to approximately 10⁶ cells per well. Each kit includes:

• MTT
• Sodium dodecyl sulfate (SDS)
• Detailed protocol (Vybrant(R) MTT Cell Proliferation Assay Kit)

This kit provides sufficient materials for ~1000 assays using standard 96-well microplates. Numerous variations and modifications of the MTT assay have been published. In addition to dehydrogenases, MTT is reduced by glutathione S-transferase (GST). Therefore, MTT may not always be a reliable cell viability probe in cells treated with compounds that affect GST activity.

FluoReporter Blue Fluorometric Nucleic Acid Assay Kit

The FluoReporter Blue Fluorometric dsDNA Quantitation Kit (F2962) provides the protocols developed by Rago and colleagues for analyzing cellular DNA with the blue-fluorescent Hoechst 33258 nucleic acid stain. The kit enables researchers to detect ~10 ng of isolated calf thymus DNA or ~1000 mouse NIH 3T3 cells in a 200 µL sample (substantially lower levels are detectable using our CyQUANT Cell Proliferation Assay Kit described above).

With this kit, quantitation of cellular DNA is rapid, and all manipulations can be carried out in microplate wells. The cells are lysed by freezing them in distilled water, which circumvents the requirement for extraction procedures used in other Hoechst 33258 dye–based protocols. The diluted dye solution is then added to the lysed cells, and fluorescence is measured. Kit components include:

• Hoechst 33258 in DMSO/H₂O
• TNE buffer
• Detailed protocol (FluoReporter(R) Blue Fluorometric dsDNA Quantitation Kit)

Each kit provides sufficient reagents for assaying approximately 2000 samples using a fluorescence microplate reader.

CountBright Absolute Counting Beads

Flow cytometry provides a rapid method for quantitating cell characteristics, however most flow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been widely used in quantitating cell populations and disease progression and are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with flow cytometry population data (multiple-platform testing) or by adding an internal microsphere counting standard to the flow cytometry sample (single-platform testing). The single-platform method is preferred as it is technically less complicated and more accurate than multiple-platform testing. CountBright absolute counting beads (C36950) are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths and contain a known concentration of microspheres. For absolute counts, a specific volume of the microsphere suspension is added to a specific volume of sample, such that the ratio of sample volume to microsphere volume is known. The volume of sample analyzed can be calculated from the number of microsphere events and then used with cell events to determine cell concentration. In general, at least 1000 bead events should be acquired to assure a statistically significant determination of sample volume. Sufficient reagents are provided for 100 flow cytometry assays, each using 50 µL of counting beads per test.

CountBright absolute counting beads are broadly fluorescent and can be used with either a fluorescence or scatter threshold.. Fluorescence can be excited by wavelengths from UV to 635 nm; fluorescence emission can be read between 385 nm and 800 nm. The fluorescence intensity of the microspheres has been adjusted to be about 5–50 times brighter than the anticipated intensities of typically stained cells. When using a scatter threshold, the microsphere signal should be above the threshold. The microspheres can be gated by a single parameter, but a combination of parameters can be used to resolve microspheres from cells and other events.

CountBright absolute counting beads can be used with any sample type, including no-wash/lysed whole blood. The microspheres in the reagents are approximately 7 µm in diameter and have settling properties similar to lymphocytes. The accuracy of cell counts based on CountBright™ absolute counting beads depends on sample handling and the precise delivery of the volume of beads. The CountBright™ absolute counting beads must be mixed well to assure a uniform suspension of microspheres. After vortexing for 30 seconds, the microsphere suspension can be pipetted by standard techniques; however, more viscous solutions such as blood require reverse pipetting for accurate volume delivery. Cell suspensions may be diluted but should be assayed without wash steps. Other sample preparation steps that can lead to cell or microsphere loss should also be avoided. For antibody protocols, CountBright absolute counting beads should be used with reagents titered for no-wash staining.

Detection and Enumeration Assays for Microorganisms and Viruses

Detecting Bacteria, Yeast and Plankton Using Nucleic Acid Stains

We recommend our Patented SYTO nucleic acid stains (see the complete list in Table 8.3) for simple detection of the presence of bacteria, yeast, mammalian cells and other nucleic acid–containing cells (, , , ). The SYTO dyes are essentially nonfluorescent except when bound to nucleic acids, where they become highly fluorescent, often with quantum yields exceeding 0.5. Consequently, it is usually not necessary to remove unbound stains before analysis. SYTO dyes are available with blue (SYTO(R) Blue Fluorescent Nucleic Acid Stains), green (SYTO(R) Green-Fluorescent Nucleic Acid Stains), orange (SYTO(R) Orange Fluorescent Nucleic Acid Stains) or red (SYTO(R) Red Fluorescent Nucleic Acid Stains) fluorescence. The SYTO dyes rapidly penetrate the membranes of almost all cells, including bacteria and yeast. The various cell types can often be identified by their characteristic morphology or, in the case of flow cytometric applications, by their light-scattering properties. The SYTO 11 and SYTO 13 green-fluorescent nucleic acid stains (and probably most other SYTO dyes) show exceptional ability to penetrate tissues for at least 100 µm, including untreated, unfixed human brain tissue, where they were used to enumerate cells by confocal laser-scanning microscopy. Simultaneous labeling with a green-fluorescent SYTO dye and a red-fluorescent nucleic acid stain — most often propidium iodide, ethidium homodimer-1