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In This Issue
Optimize Cell Viability Assays for Multicolor Panels — LIVE/DEAD® Fixable Dead Cell Stain Sampler Kit
Detect Apoptosis with Nuclear Chromatin Staining — Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Apoptosis Kit
New Mouse Targets for Flow Cytometry — Primary Anti-mouse Monoclonal Antibody Conjugates
See all of this month's New Products for Cell & Tissue Analysis
Count Blood Cells with the Countess™ Automated Cell Counter
Buzzworthy — Analysis of Electric Pulse–induced Membrane Nanopores
Online Technical Webinars — Counting Cells & Assessing Cell Viability
The View — IP3 Receptors in Rat Neurons
Proven Performers — Antibody Conjugation Kits: Minimize Time, Maximize Yield
On the Web — Build Your Own Immunoassay, The Cell Resource Center
In the Field — Ultra-High–Throughput Cell-based Assays
Check out the latest issue of BioProbes
||Dead Cell Stains in Flow Cytometry:
A comprehensive analysis.
(Also download a Comprehensive Survey of Dead Cell Stains!)
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FEATURED NEW PRODUCTS
what it is
LIVE/DEAD® cell viability assays are used to differentially stain live and dead cells in a variety of mammalian cell types. These cells can then be fixed with formaldehyde for subsequent analysis by flow cytometry. The LIVE/DEAD® Fixable Dead Cell Stain Sampler Kit contains all 8 LIVE/DEAD® reactive dyes that are excited with laser lines ranging from UV to far red, making it easier to identify the optimal dead cell stain for use in your multicolor panels.
what it offers
- Post-fixation analysis—staining pattern preserved
- Accuracy—eliminates staining artifacts
- Flexibility—broad dye selection
how it works
LIVE/DEAD® cell viability assays employ the reaction of a fluorescent reactive dye with amines on cellular proteins. Viable cells exhibit faint staining on the cell surface, whereas the dye penetrates the interior of dead cells, resulting in at least a 50-fold increase in fluorescence compared to live cells. Because the dye reacts covalently with proteins, the staining pattern is completely preserved following sample fixation with formaldehyde.
Live-cell gating using LIVE/DEAD® Fixable Violet dye eliminates staining artifacts from analysis. In a comparison between live-cell gating using scatter (A), and live-cell gating using LIVE/DEAD® Fixable Violet dye (B), staining artifacts using scatter gating are illustrated. The significant number of dead cells in subsequent analysis using scatter (C) are noted, as compared to the use of the LIVE/DEAD® Fixable Violet dye (D) to eliminate dead cells. Reproduced with permission from Elsevier ((J Immunol Methods 313:199(2006)).
what it is
Apoptotic cells undergo many characteristic morphological and biochemical changes, including the compaction and fragmentation of nuclear chromatin, plasma membrane shrinkage, and loss of membrane asymmetry. The Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Apoptosis kit employs the combination of two Molecular Probes® proprietary dyes for fluorescence analysis of both the compacted state of nuclear chromatin and changes in plasma membrane integrity.
what it offers
- Convenience—reliable alternative to annexin V assays
- Flexibility—free up space on the popular 488 nm laser
- Accuracy—combination of two reliable assays
how it works
Vybrant® DyeCycle™ Violet stain (excitation/emission maxima ~370/440 nm) is a violet-excited, cell-permeant dye that binds to double-stranded DNA. Condensed chromatin of apoptotic cells stained with Vybrant® DyeCycle™ Violet stain appears brighter than the chromatin of normal cells. SYTOX® AADvanced™ dead cell stain (excitation/emission maxima ~546/660 nm) is a red-fluorescent, cell-impermeant dye that can only penetrate cells with damaged plasma membranes. The combination of these two dyes makes it easy to clearly distinguish normal, apoptotic, and dead-cell populations by flow cytometry.
Detection of apoptosis in Jurkat cells. Jurkat cells (human T cell leukemia) were untreated (A) or treated with 10 μM camptothecin to induce apoptosis (B) Cells were then mixed with the reagents in the Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Apoptosis Kit and analyzed by flow cytometry using 405/488 nm dual excitation. Note that the camptothecin-treated cells have a higher percentage of apoptotic cells than the basal level of apoptosis observed in the control cells. A = apoptotic cells, V = viable cells, N = necrotic cells.
|Vybrant® DyeCycle™ Violet/SYTOX® AADvanced™ Apoptosis Kit
what they are
Invitrogen now offers a broad range of antibodies with mouse reactivity for a variety of flow cytometry research applications including murine studies of T, B, and NK cells, cell adhesion, cell signaling, inflammatory responses, and MHC class I molecules. These primary anti-mouse monoclonal antibodies are conjugated to fluorescein isothiocyanate (FITC) and phycoerythrin (PE), and are validated for flow cytometry.
what they offer
- New specificities—immunology targets
- Mouse reactivity—for murine studies
how they work
These FITC- and PE- labeled antibodies are excited by 488 nm lasers, and are commonly used with the first (FL1) and second (FL2) channels on most flow cytometers. FITC has fluorescence emission at 525 nm, while PE has emission at 575 nm.
Detection of CD45.1+ mouse spleen cells. SJL cells were stained with mouse anti-mouse CD45.1-FITC, and small lymphocytes were gated and analyzed on a FACScan™ flow cytometer (BDIS, San Jose, CA).
|CD103, hamster anti-mouse, FITC||500 µg/1 mL||HMCD10301|
|CD103, hamster anti-mouse, PE||100 µg/1 mL||HMCD10304|
|CD107a (LAMP-1), rat anti-mouse, FITC||500 µg/1 mL||RMCD107A01|
|CD107b (LAMP-2), rat anti-mouse, FITC||500 µg/1 mL||RMCD107B01|
|CD154, hamster anti-mouse, FITC||500 µg/1 mL||HMCD15401|
|CD154, hamster anti-mouse, PE||100 µg/1 mL||HMCD15404|
The Countess™ Automated Cell Counter
The Countess™ Automated Cell Counter uses trypan blue staining combined with a sophisticated image analysis algorithm to provide accurate cell and viability counts in just 30 seconds. The algorithm also measures the average size of live, dead, and total cells to give you all the data you need from your cell cultures without using a hemocytometer.
Counting Blood Cells is Now Faster and Easier
The Countess™ Automated Cell Counter has also been used to accurately count blood cells including peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs), and white blood cells (WBCs) from lysed whole blood and Ficoll preparations. Because the Countess™ instrument uses trypan blue staining, the viability of WBCs is easily determined, while platelets and noncellular debris are unstained and excluded from the count. It should be noted that the Countess™ instrument does not differentiate between WBC types and that viability information is not available for RBCs.
- Download the Countess™ Blood Cell Protocol
- Learn More about the Countess™ Automated Cell Counter
|Countess™ Automated Cell Counter||each||C10227|
Countess™ Automated Cell Counter Starter Kit
|Countess™ Automated Cell Counter Lab Starter Kit
(includes 1 cell counter and 101 boxes of slides)
|Countess™ Cell Counting Chamber Slides||50 slides (100 counts)||C10228|
|Countess™ Cell Counting Chamber Slides||500 slides (1,000 counts)||C10312|
|Countess™ Cell Counting Chamber Slides||1,250 slides (2,500 counts)||C10313|
|Countess™ Cell Counting Chamber Slides||2,500 slides (5,000 counts)||C10314|
|Countess™ Cell Counting Chamber Slides||5,000 slides (10,000 counts)||C10315|
The FluxOR™ Assay Enables Analysis of Electric Pulse–induced Membrane Nanopores
Lipid nanopores can form a stable, ion channel–like conduction pathway in cell membrane.
Pakhomov, A.G. et al. (2009) Biochem Biophys Res Commun (manuscript accepted).
Can electrical pulses induce ion-selective membrane disruption?
It is widely known that electric pulses can cause the formation of pores in cell membranes; the phenomenon is the basis for electroporation, an established technique for introducing substances into cells. In their recent study, Pakhomov and colleagues describe the formation and properties of very small membrane pores—"nanopores"—following the application of nanosecond electric pulses (nsEPs).
Electrophysiological observations of nanopore behavior were performed through the use of a modified FluxOR™ assay, wherein the uptake of thallium ion (Tl+) by nsEP-treated cells was monitored via an increase in the Tl+-dependent fluorescence emission of the FluxOR™ probe.
In response to applied nsEPs, both GH3 cells (characterized by the presence of multiple endogenous ion channel types) and CHO-K1 cells (which express relatively few endogenous channel types) exhibited an increase in whole-cell conductance and inward current rectification. Nanopores are so called because the group demonstrates them to be mostly impermeable to the fluorescent probe propidium iodide, setting an effective upper pore size limit of ~1 nm. nsEP-induced nanopores were stable for many minutes, and eventually dissipated either by spontaneous resealing or by the formation of larger, propidium-permeant pores.
The group suggests that nanopores could result through a number of mechanisms in addition to the application of nsEPs, and their relatively long lifetime merits their consideration as an alternative ion-selective pathway that could impact the interpretation of other studies of transmembrane ion flow.
View bibliography reference
Learn More about The FluxOR™ Assay
|FluxOR™ Thallium Detection Kit *for 10 microplates*||1 kit||F10016|
|FluxOR™ Thallium Detection Kit *for 100 microplates*||1 kit||F10017|
|Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.
Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.
Missed our previous webinars? Find our recorded webinars here!
|Midsagittal section of rat cerebellum stained to reveal IP3 receptors and glial fibrillary acidic protein (GFAP). Purkinje neurons (green) were stained using a rabbit primary antibody targeted to the IP3 receptor, and visualized with Qdot® 525 goat F(ab')2 anti-rabbit IgG conjugate. In astrocytes (red), GFAP was stained using a mouse primary antibody targeted to GFAP, and visualized with Qdot® 655 goat F(ab')2 anti-mouse IgG conjugate. Nuclei were stained blue with Hoechst 33342. Image submitted by Tom Deerinck, University of California, San Diego.
|Qdot® 525 goat F(ab')2 anti-rabbit IgG conjugate||200 µL||Q11441MP|
|Qdot® 655 goat F(ab')2 anti-mouse IgG conjugate||200 µL||Q11021MP|
|Hoechst 33342 trihydrochloride, trihydrate *FluoroPure™ grade||100 mg||H21492|
Antibody Conjugation Kits: Minimize Time, Maximize Yield
Using a direct antibody conjugate for fluorescence imaging or flow cytometry can eliminate the noise commonly associated with nonspecific secondary antibody binding. Directly labeled antibodies also allow you to use more than one same-species antibody in a single experiment. Invitrogen offers a wide variety of antibody conjugation kits for labeling µg to mg quantities of IgG antibody with bright and photostable Alexa Fluor® dyes or biotin.
Molecular Probes® antibody labeling kits have been carefully configured to minimize hands-on time while maximizing conjugate yield. Labeled antibodies are ready in as little as 2 hours and require only ~10–20 minutes of hands-on time. Which labeling kit to use depends entirely on the amount of antibody to be labeled.
The kits use time-tested, amine-reactive labels to covalently attach a fluorophore or biotin to your antibody. The result is a strong carboxamide linkage between the label and the antibody, with exceptional stability—from months to years.
- Learn More about Invitrogen's IgG Antibody Labeling Kits
Invitrogen’s comprehensive selection of antibody labeling kits is based on the amount of antibody to be labeled.
The Cell Resource Center
The Cell Resource Center is your portal to a wide range of cellular technologies including GIBCO® cells, media, and reagents, Molecular Probes® fluorescent reagents and detection technologies, Dynal® bead-based isolation systems, drug discovery products, and services to support your research needs.
- Learn More about Cellular Technologies
While you're there, check out the new Technology Chalkboard!
Ultra-High–Throughput Cell-based Assays
In collaboration with TTP LabTech, Invitrogen scientists have validated Molecular Probes® high-content screening (HCS) cell health and cytotoxicity kits on the ultra-high–throughput laser scanning Acumen® eX3 system, a high-content imaging platform for the rapid generation of multiplexed cell-based data. The combination of robust multiparametric HCS kits with rapid, whole-well data generation enabled by the Acumen® eX3 system offers a whole new level of throughput and content for a variety of cell-based imaging applications.
Download posters from the Invitrogen/TTP LabTech Collaboration, and other posters relevant to high-content imaging and analysis.
Molecular Probes® The Handbook
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