Preserve sample fluorescence with ProLong® Diamond and SlowFade® Diamond antifade mountants

ProLong® Diamond and SlowFade® Diamond antifade reagents enable superior protection against photobleaching across the visible and IR spectra. These glycerol-based liquid mountants preserve the fluorescence of cells and tissues stained with Alexa Fluor® dyes, as well as with traditional dyes such as FITC, Cy®3, and Cy®5, and fluorescent proteins including GFP, RFP, and mCherry. Both ProLong® Diamond and SlowFade® Diamond Antifade Mountants are provided already mixed and ready to use in a dropper bottle, with or without DAPI.

ProLong® Diamond Antifade Mountant suppresses photobleaching and preserves the signals of fluorescently labeled cells for long-term storage and analysis. ProLong® reagents cure within 24 hours, forming a semi-rigid gel that does not discolor or shrink during storage, making it possible to take high-quality images weeks or months after mounting.

SlowFade® Diamond Antifade Mountant is a noncuring mountant, and samples can be viewed immediately. When coverslips are sealed with an appropriate sealant, samples can be stored for days to weeks.

Image of HeLa cells mounted with ProLong® Diamond Antifade Mountant  
HeLa cells mounted with ProLong® Diamond Antifade Mountant.
HeLa cells were fixed, permeabilized, blocked with BlockAid™ Blocking Solution, and stained with ActinGreen™ 488 ReadyProbes® Reagent, NucBlue® Live ReadyProbes® Reagent, and mouse anti–ATP synthase subunit IF1 and Alexa Fluor® 594 goat anti–mouse IgG antibodies. Stained cells were mounted using ProLong® Diamond mountant and imaged on the EVOS® FL Auto Imaging System.

Ready-to-use ReadyProbes® Cell Viability Imaging Kits

ReadyProbes® Cell Viability Imaging Kits are ready-to-use reagents designed to help you quickly and easily distinguish live cells from dead cells in your sample. Each ReadyProbes® Cell Viability Imaging Kit contains a cell-permeant, blue-fluorescent nuclear stain that can cross the plasma membrane of all cells, regardless of whether they are alive or dead, as well as a cell-impermeant, green- or red-fluorescent nuclear stain that can only cross the plasma membrane once it has been compromised. Just add 2 drops of each reagent to 1 mL of your cell sample in growth medium, incubate for 5–20 minutes, and count the live blue-fluorescent cells and membrane-compromised green- or red-fluorescent cells.

ReadyProbes® reagents are formulated for maximum convenience without sacrificing any of their optical or physicochemical properties. These reagents are provided with concise protocols, convenient dropper bottles, and room temperature–stable formulations.

Image showing detection of cell viability using the ReadyProbes® Cell Viability Imaging Kit  
Detection of cell viability using the ReadyProbes® Cell Viability Imaging Kit.
A 1:1 mixture of live and heat-killed Jurkat cells was stained using the ReadyProbes® Cell Viability Imaging Kit (Blue/Green). After a 20 min incubation at room temperature, cells were imaged on the EVOS® FL Auto Imaging System; live cells fluoresce blue, and dead cells fluoresce green.

Label cells with the far-red–fluorescent CellTracker™ Deep Red Dye

For monitoring cell movement or cell location through generations, we offer CellTracker™ Deep Red Dye. This far-red–fluorescent tracer is designed to freely pass through cell membranes into live cells, where it is converted to a membrane-impermeant reaction product that is retained through cell division. Like our other CellTracker™ dyes, this dye is transferred to daughter cells but not to adjacent cells in a population, and it has been shown to not affect cell viability or proliferation at working concentrations.

CellTracker™ Deep Red Dye exhibits long-wavelength fluorescence (excitation/emission maxima ~630/650 nm) for at least 72 hours at physiological pH. Additionally, the fluorescence spectra of CellTracker™ Deep Red Dye are well separated from those of green- and red-fluorescent probes, including GFP and RFP, making it ideal for multicolor experiments.

Image showing fixed-cell multiplexing with CellTracker™ Deep Red Dye  
Fixed-cell multiplexing with CellTracker™ Deep Red Dye.
HeLa cells treated with CellTracker™ Deep Red Dye (pink), CellLight® Mitochondria-GFP (green), rabbit anti-vimentin and Alexa Fluor® 750 goat anti–rabbit IgG (white) antibodies, and NucBlue® Fixed Cell ReadyProbes® Reagent (blue).

Simplify western blot detection with WesternDot® fluorescent secondary antibody conjugates

The WesternDot® fluorescent secondary antibody conjugates, powered by Qdot® VIVID® technology, allow researchers to simplify western blot detection without sacrificing performance. Users can obtain results equivalent to those from enhanced chemiluminescence (ECL) using standard UV light boxes and gel imagers equipped with ethidium bromide or SYBR® Green detection filters. WesternDot® antibody conjugates eliminate the need for film, developer solutions, darkrooms, and enzyme optimization, and the fluorescent signals are stable over time.

Converting from ECL to WesternDot® staining is simple and straightforward. Each vial of WesternDot® antibody conjugate contains enough reagent to stain 25 miniblots when used at the recommended 1:500 dilution. WesternDot® antibody conjugates are compatible with common blocking reagents such as casein and powdered milk. To detect target proteins labeled with WesternDot® fluorescent secondary antibody conjugates, simply image the blot the same way that you would image a gel stained with ethidium bromide or SYBR® Green dyes. Unlike chemiluminescent signals, which fade over time, WesternDot® signals are extremely stable and can last for days to months.

Image showing comparison of enhanced chemiluminescence (ECL) and WesternDot® fluorescence for western blot protein detection.   Comparison of enhanced chemiluminescence (ECL) and WesternDot® fluorescence for western blot protein detection. Serial dilutions (10–0.02 µg) of Jurkat cell lysate were run on NuPAGE® Novex® 4–12% Bis-Tris precast gels and transferred to iBlot® nitrocellulose membranes using the iBlot® Gel Transfer Device. The membranes were probed with mouse anti-GAPDH antibodies followed by either fluorescence detection using WesternDot® 625 goat anti–mouse IgG (A–F) or ECL detection using a horseradish peroxidase goat anti–mouse IgG (G). WesternDot® images were collected using multiple imagers. Filter settings for fluorescence detection on each imager are as follows: A, Qdot® 625 filter; B, 645AF20 filter; C, LPG filter; D, ethidium bromide filter; E, ethidium bromide filter; F, iPhone® 5 camera and UVP Mini Benchtop UV Transilluminator. The ECL image (G) was acquired using the Fujifilm® LAS-4000 Imager.

Detect low-abundance targets with tyramide signal amplification (TSA)

For ultimate detection sensitivity, tyramide signal amplification (TSA) techniques are widely used in fluorescent immunodetection and FISH procedures to visualize targets with dim signals or low expression levels. TSA is an enzyme-mediated detection method that uses HRP to generate high-density labeling of a target protein or nucleic acid in situ. A tyramide conjugate is incubated in the presence of HRP (often bound to a secondary antibody), which catalyzes the binding of the tyramide and associated label to adjacent tyrosines for enhanced fluorescence detection (if the tyramide is conjugated to a fluorescent dye) or colorimetric detection (if the tyramide is conjugated to biotin).

TSA signals can be 100-fold stronger than those from traditional immunodetection techniques, and TSA methods can be used with both fluorogenic and chromogenic substrates on slide-based assays. Our stand-alone Alexa Fluor® and biotin tyramide conjugates are designed to work with any HRP-conjugated secondary antibody and come with the necessary amplification buffer. Also available are complete TSA kits for several common antibody species.

Image showing detection of zebrafish α-tubulin after signal amplification with TSA  
Sensitive detection of zebrafish α-tubulin.
A zebrafish cryosection was incubated with the biotin-XX conjugate of mouse monoclonal anti–α-tubulin antibody. The signal was amplified with TSA™ Kit #22, which includes HRP–streptavidin and Alexa Fluor® 488 tyramide (green). The sample was then incubated with a mouse monoclonal FRet 6 antibody and was visualized with Alexa Fluor® 647 goat anti–mouse IgG antibody (pseudocolored magenta). Finally, the nuclei were counterstained with SYTOX® Orange Nucleic Acid Stain (orange).

Fluo-4 now in a complete kit for calcium imaging applications

Fluo-4 Calcium Imaging Kit

The Fluo-4 Calcium Imaging Kit has been designed for the specific detection of calcium flux in imaging applications. Upon binding calcium, the fluo-4 dye exhibits a large fluorescence emission increase (>100-fold) with a minimal shift in wavelength maximum; the emission from calcium-bound fluo-4 dye (excitation/emission maxima ~494/506 nm) can be detected using standard fluorescein (FITC) filters.

To save you time, we have collected all the reagents you need for detecting calcium flux in live cells with the fluo-4 dye and packaged them into the Fluo-4 Calcium Imaging Kit. In addition to fluo-4 AM, which is supplied as a 1,000X solution in DMSO, this kit contains 100X PowerLoad™ Concentrate for easy cell loading, a 10X Neuro Background Suppressor, and probenecid. The Fluo-4 Calcium Imaging Kit has been formulated, optimized, and validated for imaging applications.

Image showing visualization of calcium ion flux with the Fluo-4 Calcium Imaging Kit  

 

Calcium ion flux visualized. Cytosolic calcium flux from neuronal cells detected using the Fluo-4 Calcium Imaging Kit.

 

The next generation of membrane potential probes has arrived

FluoVolt™ Membrane Potential Kit

The FluoVolt™ membrane potential dye represents the next generation in voltage-sensitive fluorescent probes, displaying the best characteristics of the fast- and slow-response membrane potential probes. The FluoVolt™ probe not only responds to changes in membrane potential in less than a millisecond like the fast-response probes, but also displays a high-magnitude fluorescence response like the slow-response probes.

The FluoVolt™ membrane potential dye is a fast-response probe with a superior potential-dependent fluorescence response. The response is fast enough to detect transient (millisecond) potential changes in excitable cells, and the probe generates a signal change in excess of 25% per 100 mV. Compatible with standard fluorescein (FITC) settings, the FluoVolt™ membrane potential dye can be used for imaging electrical activity of intact heart tissues, mapping membrane potentials along neurons and muscle fibers, or measuring potential changes in response to pharmacological stimuli.

Four panel figure showing membrane voltage changes measured using the FluoVolt™ Membrane Potential Kit

Membrane voltage changes measured using the FluoVolt™ Membrane Potential Kit. In panels A and B, differentiated NG-108 cells (mouse neuroblastoma–rat glioma hybrid) were loaded with the FluoVolt™ membrane potential dye. (A) Cells were imaged with 10 msec illumination pulses and images acquired with 2x binning. The three selected traces (B) show fluorogenic responses from the dye as the selected cells (numbered dots in A) spontaneously depolarized and repolarized in culture. For the traces in panels C and D, human HEK 293 (embryonic kidney) cells were loaded with the FluoVolt™ membrane potential dye, imaged with 10 msec illumination pulses, and data acquired with 2x binning. Traces show fluorogenic responses as cells are depolarized at 2 sec intervals from –100 mV to +30 mV (C) or in single steps from –80 mV to 0 mV at 2 sec intervals (D).

Express almost any gene using BacMam technology

ViraPower™ BacMam Expression System

The ViraPower™ BacMam Expression System provides a convenient means of expressing almost any gene of interest in mammalian cells with the use of BacMam technology. The BacMam system allows expression of genes up to 38 kb, making it possible to clone and express most genes with introns and then let the cell do the RNA processing and posttranslational modification. Among all the methods available for transfection, the BacMam gene delivery and expression system produces the least cellular toxicity. And because expression is transient and baculoviruses do not replicate in mammalian cells, special containment measures are not required.

The BacMam pCMV-Dest Vector, included as part of the ViraPower™ BacMam Expression System, combines Gateway® cloning and BacMam gene expression technologies for easy recombination-based cloning and baculovirus-based expression of a target gene in variety of cell types. In BacMam technology, a modified insect cell virus (baculovirus) is used as a vehicle to efficiently deliver and transiently express genes in mammalian cells with minimum effort and toxicity. This technology allows for control over the level of expression through the increase or decrease of viral particle concentration. When BacMam technology is combined with Gateway® cloning technology, a variety of sizes of target genes can be cloned and expressed. Sizes ranging from an insert for RNAi to a large gene with introns (38 kb) have been cloned and expressed successfully using the BacMam pCMV-Dest Vector. This plasmid accommodates various cloning schemes, including Gateway®, GeneArt® Seamless Cloning and Assembly, and traditional cloning using restriction enzymes.

Illustration of the mechanism of gene delivery using BacMam technology  

 

BacMam-mediated gene delivery. BacMam particles are taken up by endocytosis and their contents released for transcription and expression (following migration to the nucleus). Gene expression begins within 4 to 6 hr of transduction and nears its maximum level within 24 hr of transduction.


Isolate intact exosomes from a variety of starting samples

Total Exosome Isolation Reagents

With a variety of starting samples, you can now easily enrich for intact exosomes using a method that is much easier and less tedious than ultracentrifugation. We provide five Total Exosome Isolation Reagents, each optimized for enriching exosomes from cell culture media, serum, plasma, urine, or other body fluids. Their simple and reliable protocols can be scaled to a range of sample sizes and typically require only 15–20 minutes of hands-on time.

The flexible and scalable Total Exosome Isolation Reagents allow for fast and efficient exosome enrichment. After your sample is incubated with the Total Exosome Isolation Reagent overnight at 2–8°C, exosomes can be recovered using a standard benchtop centrifuge to spin them at 10,000 x g for 60 minutes. Simply resuspend the pellet, and the exosomes are ready for downstream analysis.

Two-panel line graphs showing analysis of exosomes recovered from HeLa cell media    
     
Analysis of exosomes recovered from HeLa cell media. (A) Exosomes recovered with Total Exosome Isolation Reagent (from cell culture media) have a size distribution comparable to (B) exosomes isolated following a traditional sucrose gradient ultracentrifugation protocol. Profiles analyzed on a NanoSight® LM10 instrument show all particles to be smaller than 300 nm; most are about 50–150 nm in size.
For Research Use Only. Not for use in diagnostic procedures.