In This Issue


pHrodo™ Bioparticles® Conjugates   View Phagocytosis in Green and Red—pHrodo™ BioParticles® Conjugates
Lipid Peroxidation in Live or Fixed Cells   Study Lipid Peroxidation in Fixed Cells—Click-iT® LAA Reagent and Kit
ABfinity™ Recombinant Rabbit Antibodies   Detect Retinoblastoma—New Rb ABfinity™ Recombinant Rabbit Antibodies
BacMam-Based Live-Cell Markers   Add, Incubate, and Image—Trial-Size Markers for Cell Cycle and Function
Conjugates for Flow Cytometry   New PE-Cy®7 Conjugates for Flow Cytometry



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BioProbes® Journal of Cell Biology Applications
BioProbes 67  
The Molecular Probes® Handbook
Molecular Probes Handbook


View Phagocytosis in Green and Red—pHrodo™ BioParticles® Conjugates

what they are
Proprietary, pH-sensitive pHrodo™ conjugates of E. coli, S. aureus, and zymosan are designed to be specific sensors of phagocytosis. In addition to red-fluorescent versions, we now offer green-fluorescent pHrodo™ BioParticles® conjugates that can be visualized using standard fluorescein filters.

what they offer

  • Optimized, fixable probes for detecting phagocytosis in live cells
  • Available as E. coli, S. aureus, and zymosan conjugates of pHrodo™ dye in green and red
  • Compatible with live-cell fluorescence imaging, high-content screening (HCS), flow cytometry, high-throughput screening (HTS), and benchtop instruments such as the Tali®, FLoid®, and Attune® systems

how they work
During phagocytosis, cells ingest the pHrodo™ dye-conjugated BioParticles and form phagosomes. These phagosomes fuse with the early lysosome to form the acidic phagolysosome. The nonfluorescent pHrodo™ dye, conjugated to the surface of the BioParticles® reagents, becomes fluorescent with this reduction in pH, making these particles ideal reagents for the study of phagocytosis and its regulation by drugs and environmental factors. Because pHrodo™ dye is minimally fluorescent at neutral pH, wash steps and quencher dyes are not required. To facilitate multiplexing, cells assayed for phagocytic activity with pHrodo™ BioParticles® conjugates can be fixed, preserving the fluorescent signal potentially for up to 24 hours.

Phagocytosis in MMM cells   Phagocytosis in MMM cells visualized using pHrodo™ Green E. coli BioParticles® Conjugate and pHrodo™ Red E. coli BioParticles® Conjugate. MMM cells were plated in complete medium and cultured overnight. Cells were then rinsed with 1X Live Cell Imaging Solution and treated with a suspension of pHrodo™ Red E. coli BioParticles® Conjugate (left) and pHrodo™ Green E. coli BioParticles® Conjugate (right) in Live Cell Imaging Solution at 1 mg/mL. Cells were incubated for 90 min at 37°C. Nuclei were stained with NucBlue® Live Cell Stain.
Product Cat. No. Quantity Ex/Em maxima (nm)
pHrodo™ Red E. coli BioParticles® Conjugate P35361 5 x 2 mg 560/585
pHrodo™ Green E. coli BioParticles® Conjugate P35366 5 x 2 mg 509/533
pHrodo™ Red S. aureus BioParticles® Conjugate A10010 5 x 2 mg 560/585
pHrodo™ Green S. aureus BioParticles® Conjugate P35367 5 x 2 mg 509/533
pHrodo™ Red Zymosan BioParticles® Conjugate P35364 5 x 2 mg 560/585
pHrodo™ Green Zymosan BioParticles® Conjugate P35365 5 x 2 mg 509/533

Study Lipid Peroxidation in Fixed Cells—Click-iT® LAA Reagent and Kit

what they are
The Click-iT® Lipid Peroxidation Imaging Kit – Alexa Fluor® 488 and the stand-alone Click-iT® LAA reagent employ click chemistry and linoleamide alkyne (LAA) reagent (alkyne-modified linoleic acid) to detect lipid peroxidation–derived protein modifications. The kit provides the tools needed to visualize lipid peroxidation using green-fluorescent Alexa Fluor® 488 azide, and the stand-alone reagent can be combined with a variety of azide-modified detection reagents and other tools.

what they offer

  • Easy to use—simple workflow to detect lipid peroxidation in fixed cells
  • Flexibility—choose between a kit or stand-alone reagent
  • Compatibility—use in fluorescence imaging, high content screening (HCS), flow cytometry, high-throughput screening (HTS), and with benchtop instruments such as the Tali®, FLoid®, and Attune® systems

how they work
When incubated with cells, Click-iT® LAA incorporates into cellular membranes. Upon lipid peroxidation, LAA is oxidized and produces 9- and 13-hydroperoxy-octadecadienoic acid (HPODE). These hydroperoxides decompose to multiple α,β-unsaturated aldehydes, which readily modify proteins at nucleophilic side chains. These alkyne-containing modified proteins can subsequently be detected using Click-iT® chemistry and multiplexed with other probes appropriate for fixed cells. Click-iT® LAA reagent can be detected with haptens or with azide-containing fluorophores such as the bright, photostable Alexa Fluor® azide reagents. The Click-iT® Cell Buffer Kit provides researchers with the tools required to perform the click reaction with these reagents.

Lipid peroxidation detection   Lipid peroxidation detection in bovine pulmonary artery endothelial (BPAE) cells. Cells were plated on 35 mm glass bottom dishes and fed 50 µM linoleic acid alkyne in complete growth medium at 37°C for 30 min. Cells were treated with hemin to induce lipid peroxidation–derived protein modifications (right), or left untreated (left) for 2 hr at 37°C, then fixed and permeabilized. The click reaction was performed with 5 µM Alexa Fluor® 488 azide from the Click-iT® Lipid Peroxidation Kit – Alexa Fluor® 488 for 30 min. Cells were stained with NucBlue® Live Cell Stain and imaged on a Zeiss Axiovert inverted microscope using a 40x objective.

Detect Retinoblastoma—New Rb ABfinity™ Recombinant Rabbit Antibodies

what they are
Rb, or retinoblastoma, is a tumor suppressor protein in the pocket protein family. A nuclear protein that is widely expressed in retinal cells, pRb prevents the replication of damaged DNA by preventing progression through the cell cycle from G1 to S. pRb is inactivated by phosphorylation and activated by dephosphorylation. Active (dephosphorylated) pRb binds and inactivates the cellular transcription factor E2F1, the function of which is required for cell cycle progression. Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Phosphorylation of serine 249 and threonine 252 is catalyzed by cyclin D-cdk4. We now offer ABfinity™ recombinant rabbit antibodies specific for Rb.

what they offer

  • Consistent lot-to-lot performance
  • Minimize the need to revalidate working antibody dilutions for your experiments each time you order

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. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies, combining the improved signal strength of a polyclonal antibody with the highly reproducible results you get from ABfinity™ monoclonal antibodies.

U2OS cells stained with Rb [pS780] ABfinity™ Recombinant Rabbit Oligoclonal Antibody  
Immunocytochemistry analysis of U2OS cells stained with Rb [pS780] ABfinity™ Recombinant Rabbit Oligoclonal Antibody. (A) Alexa Fluor® 488 goat anti–rabbit IgG was used as a secondary antibody (green). (B) Nuclei were stained with DAPI (blue). (C) Actin was stained with Alexa Fluor® 594 phalloidin (red). (D) Composite image of cells showing nuclear localization of phosphorylated Rb.

Add, Incubate, and Image—Trial-Size Markers for Cell Cycle and Function

what they are
Our popular Premo™ FUCCI and CellLight® reagents use BacMam technology to provide highly efficient and transient fluorescent protein labeling of live cells, including neurons and stem cells. New small pack sizes make it easy to test these premier live-cell markers with your cells.

what they offer

  • High efficiency—Greater than 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
  • Speed and convenience—simply add one or more BacMam reagents to your cells, incubate overnight, and image; or store frozen, assay-ready cells for later use
  • Robustness—expression can be easily adjusted by the dose; no replication in mammalian cells; lack of observable cytopathic effect; biosafety level (BSL) 1 classification

how they work
CellLight® reagents combine the utility and selectivity of fluorescent proteins (CFP, GFP, and TagRFP) with the transduction efficiency of BacMam 2.0, enabling unambiguous staining of organelles and cellular structures in live mammalian cells. Premo™ FUCCI Cell Cycle Sensor is a fluorescent, two-color sensor of cell cycle progression and division in live cells. Cells change from red in the G1 phase, to yellow in G1/S interphase, to green in the S, G2, and M phases. Both reagents are easy to use—simply add, incubate, and image.

Cell cycle imaging with Premo™ FUCCI Cell Cycle Sensor  
Cell cycle imaging with Premo™ FUCCI Cell Cycle Sensor. The cell cycle phases of dividing U2OS cells were visualized for 16 hr using the Premo™ FUCCI Cell Cycle Sensor. The cell in the center of the image transitions from G2/M (green), to G1 (red), and finally to S phase (yellow). After about 6 hr, the cell at the top of the image migrates down before undergoing mitosis (green) and progressing into G1 phase (red).

New PE-Cy®7 Conjugates for Flow Cytometry

what they are
Fifteen markers (twelve anti-mouse, two anti-human, and one anti-mouse/rat) are now available as R-phycoerythrin (PE)-Cy®7 conjugates.

what they offer

  • Flexibility—PE-Cy®7 conjugates can be used with the 488, 532, or 561 nm lasers
  • Convenience—packaged in smaller sizes
  • Affordability—average price is 50% less than other antibodies

how they work
PE-Cy®7 tandem conjugates allow simultaneous multicolor labeling and detection of multiple targets with excitation by the 488, 532, or 561 nm laser.

BALB/c bone marrow cells  
BALB/c bone marrow cells stained with Ly-6G/GR-1 Rat Anti-Mouse (clone RB6-8C5), PE-Cy®7 Conjugate and analyzed by flow cytometry.



Investigate Oxidative Stress and Autophagy in Live Cells

Aberrant generation of reactive oxygen species (ROS) through oxidative stress causes the disruption of normal cell mechanisms, and may lead to cell death. Although the role of autophagy in oxidative stress–induced cell death is not fully understood, the segregation and delivery of cytoplasmic cargo for degradation play a vital role in the cell survival response during early stage, ROS-induced stress. The LC3B protein generally resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the autophagosome to sequester oxidized or dysfunctional intracellular components for downstream degradation. This autophagosome ultimately fuses with the lysosome to form a structure known as the autolysosome for final degradation of damaged cytosolic materials. Life Technologies offers a wide variety of reagents for the investigation of oxidative stress and autophagy in live-cell labeling and detection experimentation.


oxidative stress and autophagy   Imaging oxidative stress and autophagy with live-cell detection reagents. Human osteosarcoma (U2OS) cells were transduced with the Premo™ Autophagy LC3B-RFP Sensor (orange) and CellLight® Lysosomes-GFP (green) and incubated for 24 hr prior to a 2-hr drug treatment with 200 µM tBHP to induce oxidative stress. A stain solution containing 5 µM CellROX® Deep Red Reagent (magenta) and 2 drops/mL of NucBlue™ Live Cell Stain (blue) was applied for 30 min at 37°C. Cells were washed and imaged with Live Cell Imaging Solution immediately after labeling. The sequestering and degradation of oxidized materials is evident where orange LC3B-RFP and green lysosomes-GFP localize to form the autolysosomal membranes surrounding magenta-colored cellular constituents stained with CellROX® Deep Red Reagent.




Imaging Corner

Imaging Neonatal Human Dermal Fibroblasts

Neonatal human dermal fibroblasts (HDFn) were grown on coverslips, then fixed and permeabilized using the Image-iT® Fixation/Permeabilization Kit. Cells were probed with Golgin-97 Mouse Anti-Human mAb and visualized with Alexa Fluor® 488 Goat Anti–Mouse IgG Antibody (green). Actin filaments were labeled with Alexa Fluor® 594 Phalloidin (red), and nuclei were stained with NucBlue™ Fixed Cell Stain (blue). Coverslips were mounted on slides using ProLong® Gold Antifade Reagent and imaged at 60x magnification. Image contributed by Michelle Yan, Life Technologies.


From the Bench

Detecting Protein Synthesis in Vivo Is Just a Click Away

Hinz FI, Dieterich DC, Tirrell DA, Schuman EM. (2012) Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem Neurosci 18:40–49.

Studies in a variety of different model organisms have shown that new protein synthesis is required for long-term synaptic changes and the formation of long-term memory. Modified amino acids, such as our Click-iT® AHA (L-azidohomoalanine) and Click-iT® HPG (L-homopropargylglycine) can be incorporated into proteins during active synthesis and labeled in a highly specific azide–alkyne click reaction to identify nascent proteins without using radioactivity. This technique has previously been used to examine new protein synthesis in vitro. However, Schuman and colleagues have recently labeled newly synthesized proteins in live zebrafish using Click-iT® AHA. After in vivo incorporation of Click-iT® AHA, zebrafish lysates were labeled with biotin alkyne, and biotinylated proteins were quantified and detected using dot blots. Using an Alexa Fluor® 488 alkyne, the authors were also able to image a whole-mount zebrafish to localize areas of new protein synthesis within the intact organism. The team further showed that in vivo labeling of nascent proteins with Click-iT® AHA did not affect several zebrafish behaviors and that induction of seizures results in an increase in protein synthesis that can be detected by Click-iT® AHA and the click labeling reagents. Based on their findings, the Click-iT® AHA and HPG amino acids could prove useful in examining new protein synthesis that occurs in live animals during memory formation as well as determining the specific proteins that are involved in long-term synaptic changes underlying these behaviors.

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