In This Issue
FEATURED NEW PRODUCTS
No Better Time to React—Alexa Fluor® Amine-Reactive Labels Simplified Cell Cycle Analysis of Fixed Cells—FxCycle™ PI/RNase Staining Solution Quick and Easy Cell Cycle Analysis at Your Benchtop—Tali® Cell Cycle Kit ABfinity™ Recombinant Antibodies for TIMP4 New Apoptosis Assay for Flow Cytometry—CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit See all of this month's New Antibodies
The Ballad of Technical Support
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|BioProbes® Journal of Cell Biology Applications|
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No Better Time to React—Alexa Fluor® Amine-Reactive Labels
What They Are
You can conjugate these amine-reactive reagents with virtually any protein or peptide for subsequent use in fluorescence microscopy, high-content screening, and flow cytometry applications.
New sizes of the popular Alexa Fluor® amine-reactive dyes offer added convenience when performing small- or large-scale conjugations. The 3 x 100 µg size is ideal for optimizing reaction conditions, because it provides Alexa Fluor® amine-reactive dyes in single-use quantities, eliminating the need to perform multiple conjugations simultaneously or realiquot a larger unit size and risk losing material in the process. For large-scale reactions, the new larger unit size eliminates the need to combine multiple vials.
What They Offer
- Convenience—lets you focus on the reaction, not dye handling, with optimized unit sizes
- Brightness—superior fluorescence intensity compared to other spectrally similar fluorophores
- Photostability—allows more time for image capture
How They Work
Alexa Fluor® dyes are reactive molecules that can be used to add a fluorescent label to primary amines of proteins, amine-modified oligonucleotides, and other amine-containing molecules.
- Learn more about Alexa Fluor® amine-reactive labels
Detecting stem cell surface markers using fluorescently labeled IgM antibodies. Tra-1-81 antibodies were labeled using (A) Alexa Fluor® 488 SDP ester, (B) Alexa Fluor® 594 NHS ester, and (C) Alexa Fluor® 647 NHS ester. H9 human embryonic stem cells on a feeder layer of irradiated murine embryonic fibroblasts were cultured on hESC medium containing 20% KSR and 4 ng/mL bFGF. Cells were stained with dye-conjugated antibodies at 5 µg/mL, and images were collected on a Zeiss Axiovert® fluorescence microscope at 10x magnification.
|Alexa Fluor® 488 SDP Ester||3 x 100 µg||A37570|
|Alexa Fluor® 555 NHS Ester||3 x 100 µg||A37571|
|Alexa Fluor® 594 NHS Ester||3 x 100 µg||A37572|
|Alexa Fluor® 647 NHS Ester||3 x 100 µg||A37573|
|Alexa Fluor® 680 NHS Ester||3 x 100 µg||A37574|
|Alexa Fluor® 750 NHS Ester||3 x 100 µg||A37575|
What It Is
FxCycle™ PI/RNase Staining Solution is used for flow cytometric analysis of DNA content in fixed cells. The staining solution comes ready to use, formulated with DNase-free RNase A and a permeabilization reagent in Dulbecco’s phosphate-buffered saline; just add the solution to fixed cells, incubate, and acquire data on a flow cytometer.
What It Offers
- Ready-to-use solution—preformulated solution of propidium iodide (PI) with DNAse-free RNase A
- Simple protocol—add staining solution to fixed cells, incubate at room temperature, and acquire data; no need for washing steps
- Stability—store and use at room temperature
How It Works
PI, a popular red-fluorescent stain, binds to DNA by intercalating between the bases with little or no sequence preference and a stoichiometry of one dye molecule per 4–5 base pairs. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining. FxCycle™ PI/RNase Staining Solution includes both the dye and RNase.
- Learn more about FxCycle™ PI/RNase Staining Solution
Histogram of Jurkat cells stained with FxCycle™ PI/RNase Staining Solution showing DNA content distribution. Jurkat cells were fixed in 70% ethanol, washed, and resuspended in FxCycle™ PI/RNase Staining Solution for 30 minutes at room temperature. G0/G1 and G2/M phase peaks are separated by the S phase distribution. Flow cytometric analysis was performed using 532 nm excitation with a 585/42 nm bandpass filter.
What It Is
The Tali® Cell Cycle Kit provides a room temperature–stable, ready-to-use solution for convenient and accurate determination of the percentage of cells in each phase of the cell cycle. This kit contains a single solution composed of propidium iodide (PI), RNase A, and Triton® X-100 in buffer at optimized concentrations.
What It Offers
- An all-in-one solution—contains everything you need to stain cells for cell cycle analysis
- Simple protocol—fix your cells, add reagent, incubate, and visualize
- Flexibility in analysis of results—data can be analyzed on the Tali® instrument or easily exported for in-depth analysis using modeling software of your choice
How It Works
PI is a red-fluorescent molecule that binds DNA and RNA by intercalating between the bases with little or no sequence preference; the binding is proportional to the amount of these nucleic acids, so PI can be used to accurately determine the amount of cellular DNA for cell cycle studies after treating samples with RNase. The Tali® Cell Cycle Kit includes both PI and RNase in a single solution.
- Learn more about the Tali® Cell Cycle Kit
Cell cycle histogram obtained using the Tali® Cell Cycle Kit and the Tali® Image-Based Cytometer. Jurkat cells were fixed with 70% ethanol, then stained with 200 µL of the Tali® Cell Cycle Kit solution. After incubation for 30 minutes, the samples were analyzed on the Tali® Image-Based Cytometer using the “Cell Cycle” assay. The resulting histogram has the expected distribution for normal cycling cells.
|Tali® Cell Cycle Kit||1 kit||A10798|
|Tali® Image-Based Cytometer||1 instrument||T10796|
|Tali® Cellular Analysis Slides||50 slides||T10794|
What They Are
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order. Over the past year, we have launched several new ABfinity™ recombinant antibodies.
Our latest ABfinity™ antibodies are to tissue inhibitor of metalloproteinase 4 (TIMP4). TIMP4 functions to inhibit the activity of matrix metalloproteinases, which degrade components of the extracellular matrix. TIMP4 binds irreversibly to zinc-dependent matrix metalloproteinases and inactivates them by binding to their catalytic zinc cofactor. Other functions of TIMP4 include nervous system development and platelet recruitment and aggregation.
What They Offer
- Specificity—antibodies undergo rigorous validation
- High performance—proven consistency from lot to lot
- Efficiency—detection of low-level targets with less sample
How They Work
ABfinity™ antibodies are manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain antibody cDNA. This production process offers consistent lot-to-lot antibody performance.
ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. They combine the improved signal strength that can come from using a polyclonal antibody with the highly reproducible results obtained using ABfinity™ monoclonal antibodies.
- Learn more about ABfinity™ recombinant antibodies
- Find new antibodies every month
- Search for antibodies
Immunocytochemistry analysis of purified ABfinity™ recombinant rabbit anti–human TIMP4 antibody. Immunocytochemistry analysis of U2OS cells stained with the ABfinity™ recombinant TIMP4 antibody. (A) Alexa Fluor® 488 goat anti-rabbit was used as secondary antibody (green). (B) DAPI was used to stain the nucleus (blue) and (C) Alexa Fluor® 594 phalloidin was used to stain actin (red). (D) Composite image of cells showing cytoplasmic localization of TIMP4.
|TIMP4 Monoclonal Antibody (clone 13H4L13), ABfinity™ Recombinant||100 µg||701317|
|TIMP4 Oligoclonal Antibody (clone 13HCLC), ABfinity™ Recombinant||100 µg||710340|
What It Is
The CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit is designed for detection of activated caspase-3 and -7 in apoptotic cells. This kit allows for easy discrimination of apoptotic cells from live and necrotic cells by flow cytometry.
What It Offers
- Quick and easy identification of cell populations—washing and fixation are not required
- Specificity—includes the recognition sequences for activated caspase-3 and caspase-7
- Multicolor compatibility—combine with other dyes to increase the number of detectable parameters
How It Works
This cell-permeant CellEvent® reagent consists of a 4–amino acid peptide, DEVD, conjugated to a nucleic acid–binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and cleave the caspase-3/7 recognition sequence in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels apoptotic cells with a bright, fluorogenic signal. The SYTOX® AADvanced™ Dead Cell Stain in the kit allows discrimination of apoptotic cells from live and necrotic cells. The fluorescent signals from live, apoptotic, and necrotic cells can be easily distinguished by flow cytometry.
Learn more about the CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit
Jurkat cells (T cell leukemia, human) were treated for 3 hours with (A) DMSO or (B) 10 μM camptothecin before labeling with the CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit. Stained samples were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser, and fluorescence emission was collected using a 530/30BP filter for CellEvent® Caspase-3/7 Green Detection Reagent and a 690/50BP filter for SYTOX® AADvanced™ stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, V = viable cells, N = necrotic cells.
|CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit
Improve Phosphoprotein Detection Using the Attune® Acoustic Focusing Cytometer
The study of mitogen-activated protein kinase (MAPK) is important for investigating diseases such as cancer. MAPK signaling cascades play important roles in critical decision processes within the cell, including cellular responses to environmental stimuli and disease progression.
Multiparameter flow cytometry is a valuable tool for dissecting signaling pathways in cell populations using intracellular staining with fluorescent antibodies against phosphorylation site–specific proteins. While there are reagents and techniques available for phosphoprotein-specific detection, the signals that result from these experiments are usually dim and difficult to distinguish. Advancements in instrumentation using acoustic focusing—the Attune® Acoustic Focusing Cytometer— enable better detection of dim signals than by conventional hydrodynamic focusing. The Attune® cytometer employs high-frequency sound waves to maintain a tightly focused sample stream, allowing greater precision at the laser interrogation point. Using the High Sensitive transit time setting to slow the sample stream allows longer laser interrogation time, which increases the sensitivity of detection.
We used three phosphoproteins, Akt, Erk1/2, and p38, to demonstrate an ideal research application for the Attune® cytometer, which enables the detection of dim signals through highly sensitive and precise data-gathering capabilities.
|Comparison of High Sensitive and Standard transit times (using 25 μL/min sample injection rate) on the Attune® Acoustic Focusing Cytometer to the Low Flow Rates (12 μL/min) of the BD™ LSR II and BD FACSCalibur™ instruments, using Jurkat cells treated with anisomycin and stained with p38 Alexa Fluor® 488 direct conjugate. Purple traces represent untreated, p38 Alexa Fluor® 488–stained Jurkat cells; blue traces represent anisomycin-treated, p38 Alexa Fluor® 488–stained Jurkat cells. The Attune® Acoustic Focusing Cytometer demonstrates improved separation of low-expressed proteins using the High Sensitive mode, compared to the conventional instruments using hydrodynamic focusing. (A) High Sensitive 25 μL/min, showing unstained, untreated Jurkat cells for reference (red trace), SI = 3.1; (B) High Sensitive 25 μL/min (unstained cells not shown), SI = 3.1; (C) Standard 25 μL/min (unstained cells not shown), SI = 2.7; (D) BD FACSCalibur™ 12 μL/min (Low Flow Rate) (unstained cells not shown), SI = 3.1; (E) BD™ LSR II 12 μL/min (Low Flow Rate) (unstained cells not shown), SI = 3.2.|
Videos Demonstrating Utility of pHrodo™ Red Indicator for Real-Time Imaging of Membrane Internalization and Endosome Acidification
Check out videos of cells labeled on the outer membrane and the internalization of the dye to acidic endosomes.
To capture these videos, HeLa cells were exposed to UV for 5 hours in DPBS. The supernatant was saved and the cells were labeled with pHrodo™ Red reagent, a pH-sensitive dye that increases in brightness with acidification. Mouse monocyte macrophage cells were plated on 35 mm MatTek dishes at a density of 250,000 cells per plate and labeled with NucBlue® Live Cell Stain (Hoechst 33342) to counterstain nuclei. 300,000 apoptotic HeLa cells were added to the macrophages in a 1:1 solution of Live Cell Imaging Solution and the previously saved DPBS supernatant from the UV treatment. Cells were imaged at 40x on an Applied Precision DeltaVision® system with an Olympus IX71 microscope and Photometrics CoolSNAP™ HQ2 camera, with time-lapse imaging every 45 seconds for 5 hours. Channels: DIC, DAPI, and TRITC. Images were deconvolved and zoomed. Movie speed: 10 frames/second for video 1; 7.5 frames/second for video 2.
Video 1. Mouse macrophages phagocytosing apoptotic human cervical cancer (HeLa) cells.
Video 2. Mouse macrophages phagocytosing apoptotic human cervical cancer (HeLa) cells.
|pHrodo™ Red, Succinimidyl Ester||1 mg||P36600|
|NucBlue® Live Cell Stain (Hoechst 33342 special formulation)||1 kit||R37605|
|Live Cell Imaging Solution||500 mL||A14291DJ|
Handy Page to Bookmark for New Molecular Probes® Products
Life Technologies is committed to developing new Molecular Probes® products to keep you at the forefront of life science research. Wondering what you might have missed? The new Molecular Probes® products page provides you with links to all of the products introduced over the last twelve months. Bookmark this page and check it often to see the latest products to emerge from our R&D laboratories.
CellLight® Reagents Facilitate Colocalization Studies Related to Endocytosis
Cheng J, Grassart A, Drubin DG (2012) Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis. Mol Biol Cell 23(15):2891–2904.Ghislat G, Aguado C, Knecht E (2012) Annexin A5 stimulates autophagy and inhibits endocytosis. J Cell Sci 125:92–107.
Colocalization of proteins and subcellular organelles can offer insights into cellular processes. CellLight® reagents are ready-to-use fluorescent protein constructs targeted to specific subcellular structures of mammalian cells. Several CellLight® BacMam constructs are targeted for expression in the organelles involved in the endocytosis pathway, including early endosomes, late endosomes, lysosomes, the Golgi apparatus, and the endoplasmic reticulum. Expression of these constructs allows detection and imaging of individual organelles through either Green Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP) fused to the organelle marker proteins.
This approach to organelle detection means that with a one-step protocol, researchers can specifically detect multiple organelles or proteins by selecting fluorescent dyes emitting different colored signals.
Recently, Cheng et al. used CellLight® reagents to specifically detect early endosomes in order to colocalize Alexa Fluor® 488–conjugated transferrin in SK-MEL-28 cells. Relying on these tools, the group studied transferrin trafficking and the associated effects of myosin 1E recruitment, allowing them to draw conclusions about the role of myosin 1E in regulation of actin assembly during endocytosis.
Ghislat et al. utilized CellLight® markers to colocalize annexin A5 with the Golgi complex, endoplasmic reticulum, early endosomes, and late endosomes. This enabled them to study the effects of annexin A5 on endocytosis and autophagy, and to conclude that annexin A5 regulates both the endocytic and autophagic pathways, including autophagosome fusion with lysosomes and endosomes.
Molecular Probes scientists have been providing first-in-class support for our innovative fluorescent reagents and labeling technologies for years. To share this expertise with more researchers, we have a series of free webinars focused on key fluorescence applications. Choose your learning module and get started today.
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