ProbesOnline September 2009

In This Issue

FEATURED NEW PRODUCTS

 
Accurate Compensation Settings Every TimeAbC™ anti-Rat/ Hamster Bead Kit

 
Ultrasensitive cAMP Detection  cAMP Chemiluminescent Immunoassay Kit

 
Picture-Perfect Imagery with Qdot® Nanocrystals Qmount™ Qdot® Mounting Media


 
Simultaneously Distinguish Early and Late Apoptotic CellsPacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit


New Products for Cell &Tissue Analysis
 
See all of this month's New Products for Cell & Tissue Analysis


NEW APPLICATIONS

 
Fluorescent Alternatives to Chromogenic-Based Immunohistochemistry

FEATURED NEW PRODUCTS

what it is
Accurate compensation is crucial for effective multicolor analysis in flow cytometry. Cell-based compensation controls are often not completely effective, because many antigens are not highly expressed, and dimly stained cells can lead to inaccurate compensation settings. The bead-based AbC™ anti-Rat/Hamster Bead Kit provides a method for setting accurate compensation using polystyrene microspheres either coated with immunoglobulin that reacts with all isotypes of rat and hamster Ig (positive compensation beads), or uncoated (negative compensation beads).

what it offers

  • Fast and simple bead-based compensation—an alternative to using precious samples
  • High reactivity—reacts with all subclasses of rat and hamster Ig
  • Accurate and consistent results—avoids the variations in antigen expression that often occur with cell-based assays

how it works
After incubation with the same fluorophore-conjugated rat or hamster antibody that will be used for cell staining, the beads are washed in staining buffer, a drop containing negative control beads is added, and the beads are resuspended and analyzed by flow cytometry. The two components provide distinct positive and negative populations of beads that you can use to set compensation.

 AbC™ anti-Rat/Hamster Bead Kit
Click on image for larger view

Compensation using the AbC™ anti-Rat/Hamster Bead Kit. (A) FITC-conjugated rat anti-mouse CD4 antibodies were labeled with the AbC™ RH Capture Beads for a positive signal, and the negative beads provided a negative signal. (B) Phycoerythrin-conjugated rat anti-mouse CD8a antibodies were labeled with the AbC™ Rat/Hamster Capture Beads for a positive signal, and the negative beads provided a negative signal. (C) Dual parameter plot showing gated human lymphocytes labeled with both FITC-conjugated rat anti-mouse CD4 and phycoerythrin-conjugated rat anti-mouse CD8a antibodies using compensation settings obtained with the AbC™ anti-Rat/Hamster Bead Kit.
Product Quantity Cat. No.
AbC™ anti-Rat/Hamster Bead Kit 1 kit A10389

what it is
The cAMP Chemiluminescent Immunoassay Kit enables ultrasensitive determination of cyclic AMP (cAMP) levels in cell lysates. This competitive immunoassay is formatted with maximum flexibility to permit either manual assays or automated high-throughput screening.

what it offers
Chemiluminescence detection offers substantial benefits that colorimetric, fluorometric, and isotopic methods cannot match:

  • Wide dynamic range
  • Highest sensitivity of any commercially available cAMP assay
  • Compatibility with multiple assay formats

how it works
The cAMP assay utilizes the highly sensitive chemiluminescent alkaline phosphatase (AP) substrate CSPD® with the Sapphire-II™ luminescence enhancer. The ready-to-use substrate⁄enhancer reagent generates sustained-glow light emission that is measured 30 minutes after addition.

Click on image for larger view

Cyclic AMP standard curve. cAMP standard (0.006 to 6,000 pmol) was assayed with the cAMP Chemiluminescent Immunoassay Kit. Measurements were made on the TR717™ luminometer.
Product Quantity Cat. No.
cAMP Chemiluminescent Immunoassay Kit Two 96-well plates C10557
cAMP Chemiluminescent Immunoassay Kit Ten 96-well plates C10558

what it is
Qmount™ Qdot® mounting media is a nonaqueous, permanent mountant that is critical for performing microscopy with samples labeled with Qdot® nanocrystals. Unlike other mountants, Qmount™ medium completely preserves both initial and long-term Qdot® fluorescence.

what it offers

  • Signal stability—preserves the fluorescence of Qdot® nanocrystals for months
  • Compatibility—ideal for multicolor applications with all eight Qdot® nanocrystals, their conjugates, and Qnuclear™ Deep Red stain
  • Convenience—provided ready-to-use in dropper bottles

how it works
Qmount™ Qdot® mounting media is provided as a ready-to-use solution in convenient dropper bottles. After application, Qmount™ Qdot® mounting media hardens within 12 hours, and has a refractive index of 1.5 when fully cured.

Click on image for larger view

Human carcinoma (HeLa) cell labeled with Qdot® nanocrystals. Mitochondria were detected with anti–OxPhos Complex V inhibitor protein IgG and labeled with Qdot® 625 goat F(ab')2 anti–mouse IgG conjugate (red fluorescence); the Golgi apparatus was detected with rabbit anti-giantin and labeled with Qdot® 585 goat F(ab')2 anti–rabbit IgG conjugate (yellow fluorescence); tubulin was detected with rat anti-tubulin and labeled with DSB-X™ biotin goat anti–rat IgG and Qdot® 525 streptavidin conjugate (green fluorescence). The nucleus was labeled with Qnuclear™ Deep Red stain (purple fluorescence), and the slide was mounted using Qmount™ Qdot® mounting media.
Product
Quantity Cat. No.
Qmount™ Qdot® mounting media 3 x 2 mL Q10336
     

what it is
The Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit provides a rapid and convenient flow cytometry assay for apoptosis.

what it offers

  • Multiplexing capability allows you to measure additional apoptotic parameters
  • Maximizes instrument capability by moving annexin V to the less commonly used violet laser
  • Accurate detection of early and late apoptotic cells

how it works
The kit contains recombinant annexin V conjugated to the violet-excitable Pacific Blue™ dye. The kit also includes the red-fluorescent SYTOX® AADvanced™ Dead Cell Stain for the identification of necrotic cells based on membrane integrity. After staining, apoptotic cells show bright violet fluorescence, dead cells show red fluorescence, and live cells show dim violet fluorescence. Because there is very little spectral overlap between the two dyes, very little or no compensation is required. 

Click on image for larger view

Detecting apoptosis with the Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit. Jurkat cells (human T cell leukemia) were untreated (A) or treated with 10 μM camptothecin for 4 hr (B). Cells were treated with the reagents in the Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit and analyzed by flow cytometry using 405 nm and 488 nm excitation. Note that the camptothecin-treated cells have a higher percentage of apoptotic cells than the basal level of apoptosis seen in the control cells. A = apoptotic cells, V = viable cells, N = necrotic cells.
Product Quantity Cat. No.
Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit 50 assays A35136

NEW APPLICATIONS

Towards more quantitative, multiplexed results with stable fluorescent dyes, reference beads, and superior antifade reagents

Immunohistochemistry (IHC) protocols for tissues and tissue microarrays are dominated by approaches using enzyme-coupled secondary antibodies that deposit chromogenic substrates. Their long history, well-established workflows, and archival properties support this approach. Yet standard immunohistochemistry techniques are limited to semiquantitative analysis (i.e., 0, +1, +2, +3) and narrow dynamic range, as enzymatic reactions tend to saturate, and are most often limited to one or two analytes. This is a serious complication in cancer diagnostics as the number of biomarkers increases, demanding protocols that can analyze more and more analytes with improved quantitation.

Several recent publications [1–4] show that protocols that incorporate fluorescent organic dyes, inorganic Qdot® nanocrystals, superior antifade mountants, and intensity calibration standards can replace traditional chromogenic methods. Fluorescence-based methods provide many of the advantages of traditional approaches, including ease of use and stable signal amplification, but benefit from broader dynamic ranges and the improved resolving power of direct vs. enzyme conjugates. Fluorescent labels also demonstrate an extended storage life and offer the possibility of adding 2–5 or more separate labels.

Explore Invitrogen's antibody offerings for IHC and other applications.


Fluorescent detection of E-cadherin

Fluorescent detection of E-cadherin, Ksp-cadherin, and collagen IV in kidney.
3 μm FFPE sections of adult human kidney were dewaxed, rehydrated, and subject to antigen retrieval. Sections were stained with anti–E-cadherin mAb and anti-Ksp–cadherin mAb or with anti-E-cadherin mAb and anti-collagen IV Ab followed by Alexa Fluor® 488–conjugated goat anti-mouse IgG2A and Alexa Fluor® 555 goat anti-mouse IgG1 or Alexa  Fluor®488-conjugated goat anti-mouse IgG2A and Alexa Fluor ® 555 goat anti-rabbit Ig. Nuclei were counterstained with DAPI. Arrows indicate tubules expressing both cadherins; arrowheads indicate tubules expressing neither cadherin. * Indicates tubules expressing E-cadherin alone. Scale bar, 50 μm. Reproduced from BioMed Central: Robertson D, Savage K, Reis-Filho JS, Isacke CM (2008) BMC Cell Biol 19:9.

References

1. BMC Cell Biol 19:9 (2008)
2. Cancer Epidemiol Biomarkers Prev 16:1371 (2007)
3. Nano Lett 6:2881 (2006)
4. Nat Protoc 2:1152 (2007)
Product Quantity Cat. No.
ProLong® Gold antifade reagent, special packaging 5 x 2 mL P36934
ProLong® Gold antifade reagent 10 mL P36930
ProLong® Gold antifade reagent with DAPI, special packaging 5 x 2 mL P36935
ProLong® Gold antifade reagent with DAPI 10 mL P36931
Qmount™ Qdot® Mounting Media 3 x 2 mL Q10336
InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7219
InSpeck™ Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7221
InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7223
InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7224
InSpeck™ Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm 1 kit I7225
InSpeck™ Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm 1 kit I14785
InSpeck™ Orange (540/560) Microscope Image Intensity Calibration Kit, 6 µm 1 kit I14786
InSpeck™ Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm 1 kit I14787

DEPARTMENTS

Buzzworthy

EdU-based labeling reveals differences in how cell lines react to thymidine substitution

Cell type specific applicability of 5-ethynyl-2’-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry.
Diermeier-Daucher S, Clarke ST, Hill D et al. (2009) Cytometry 75A:535–546.

Do all cell lines react the same to nucleoside analog–based labeling?
The nucleoside analog EdU (5-ethynyl-2’-deoxyuridine) offers significant advantages over BrdU for assessing the proliferation state and cell cycle status of cells, chief among them being the ability to detect the probe without denaturing the cellular DNA. However, the impact of EdU on cell viability and function has not been extensively characterized. In their current study, Diermeier-Daucher and colleagues examined the effect of pulse- and continuous labeling of two breast cancer cell lines (BT474 and SK-BR-3) with EdU as compared to the effect seen with BrdU labeling under the same conditions.

Differences in long-  and short-term EdU exposure
Each nucleoside analog yielded essentially identical staining intensity. However, incubation with EdU revealed a dose- and time-dependent increase in necrotic cell death in SK-BR-3 cells following 96 hr exposure, and both cell lines showed an increase in cell cycle arrest following long-term (144 hr) exposure to EdU. No negative impact was observed with these cells following short-pulse labeling with EdU, illustrating its utility as a sensitive and quantitative probe for flow cytometric assessment of proliferation in these cells. 

Conclusion
As previous studies have reported similar negative cellular effects associated with exposure to BrdU, the authors demonstrate the necessity of evaluating the individual response of a given cell line to nucleoside analog exposure prior to carrying out continuous labeling experiments.

View bibliography reference

Free Online Technical Webinars 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.
Webinars Date Time
Investigating Apoptosis September 29, 2009 10:00 a.m. PT


Missed our previous webinars? Find our recorded webinars here!





Qdot® nanocrystals, organic dyes, and antibodies
 

Combining Qdot® nanocrystals, organic dyes, and antibodies for superior cellular imaging.
Human carcinoma (HeLa) cells were labeled with mouse anti-OxPhos Complex V inhibitor protein IgG, Qdot® 625–conjugated goat anti-mouse IgG (red), rabbit anti-giantin (Covance Bio), Qdot® 585–conjugated goat anti-rabbit IgG (yellow), and rat anti-tubulin (Serotec). Secondary labeling was performed with anti–DSB-X biotin goat anti-rat IgG, and a tertiary streptavidin–conjugated Qdot® 525 (green). The cells were then labeled with Qnuclear™ Deep Red stain (purple) and mounted in Qmount™ Qdot® mounting media.



Tracking Stem Cells With Qdot® Nanocrystals

Tracking the migration of stem cells to target tissues and analysis of their subsequent differentiation are critical to the development of therapeutic regimes for stem cell therapy. Once differentiated, stem cells lose all stem cell–associated markers as they blend into the resident environment; they must be labeled prior to migration to be followed through differentiation. In addition to MRI, expression tags (e.g., fluorescent proteins), and organic dye–based reporters (e.g., CM-DiI, CFSE), Qdot® nanocrystals are particularly useful for stem cell labeling because they provide an extremely bright and photostable signal, even into the near infrared. Qdot® nanocrystals are detectable for at least 14 days postinjection [1], and have demonstrated up to at least 8 weeks of stability in another study [2].

Qdot® nanocrystals also offer:

  • Easy delivery to cells
  • Stability through many rounds of cell division (up to 6 or more depending on detection method and loading levels)
  • No effects on cell proliferation [2], pluripotency/differentiation, or animal physiology
  • No transfer to neighboring cells
  • Availability in many colors 


Several recent publications highlight the utility of Qdot® nanocrystals in this essential application area [1–4]. Reported delivery methods vary, with one study [2] preferring 24-hour incubations with Qdot® ITK™ carboxyl nanocrystals, but most preferring to use the cell-penetrating peptide conjugates included in the Qtracker® Cell Labeling kits [1,3,4]. Jablonski et al. [4] have shown most recently that incubation of Qdot® nanocrystals with pyrenebutyrate allows passive diffusion of poly-Arg conjugated nanocrystals into cells, resulting in a much more uniform, non-granule staining pattern. 

An important cautionary note: All Qdot®-labeled material needs to be mounted in QmountQdot® mounting media or PBS. Avoid mounting in any organic dye–based mountant such as ProLong® Gold. 

References
1. BMC Biotechnol 7:67 (2007)
2. Stem Cells 25:2128 (2007)
3. Histochem Cell Biol 130:329 (2008)
4. J Phys Chem B 113:405 (2009)

 

QTracker quantum dot

Qtracker® quantum dot label reveals distribution of injected human mesenchymal stem cells. Qtracker® 655 non-targeted quantum dot–labeled human mesenchymal stem cells were injected into a canine heart ventricle in an effort to create a biological pacemaker. Injected cells (red labeled cells in panels A and B) were detected 1 day later in fixed and sectioned material at (A) low magnification and (B) high magnification along with blue Hoechst 33342 staining. Additional experiments have shown that stem cells labeled in this manner are visible after 8 weeks and migrate in a pattern that mimics the resident in situ myocardial orientation [2]. Image courtesy of Amy Rosen, Institute for Molecular Cardiology, State University of New York at Stony Brook.

 

Product Quantity Cat. No.  
Qtracker® 625 Cell Labeling Kit1 kitA10198Order Now
Qtracker® 625 non-targeted quantum dots200 µLA10199Order Now
Qtracker® 655 non-targeted quantum dots, 2 µM solution200 µLQ21021MPOrder Now
Qtracker® 565 non-targeted quantum dots, 2 µM solution200 µLQ21031MPOrder Now
Qtracker® 705 Cell Labeling Kit1 kitQ25061MPOrder Now
Qtracker® 800 Cell Labeling Kit1 kitQ25071MPOrder Now
Qtracker® 705 non-targeted quantum dots, 2 µM solution200 µLQ21061MPOrder Now
Qtracker® 800 non-targeted quantum dots, 2 µM solution200 µLQ21071MPOrder Now
Qtracker® 605 Cell Labeling Kit1 kitQ25001MPOrder Now
Qtracker® 585 Cell Labeling Kit1 kitQ25011MPOrder Now
Qtracker® 655 Cell Labeling Kit1 kitQ25021MPOrder Now
Qtracker® 565 Cell Labeling Kit1 kitQ25031MPOrder Now
Qtracker® 525 Cell Labeling Kit1 kitQ25041MPOrder Now
Qmount™ Qdot® Mounting Media3 x 2 mLQ10336Order Now

 

Click-iT® EdU Cell Proliferation Assays New Web Resource for Click-iT® EdU Cell Proliferation Assays

Click-iT® EdU Cell Proliferation Assays offer faster, easier detection of newly synthesized DNA in a variety of fluorescent colors. Our new web page devoted to these assays allows you to select the flow cytometry, imaging, or high-content screening kit that is best for your research.

 

 

Alexa Fluor® Dyes 

New Web Page Dedicated to Alexa Fluor® Dyes

It’s now easier than ever for you to find information about Alexa Fluor® dye products. With our newly redesigned web pages, you can quickly navigate to product information, performance data, and comparisons to other dyes. You can conveniently find and order the right Alexa Fluor® product for your research needs, whether it’s a reactive dye, a secondary antibody, or other functional detection assays that employ the highest-performing dyes. With thousands of peer-reviewed references and the best technical support available,* you can feel confident that you’ll have the best research experience, regardless of which Alexa Fluor® products you choose.

* Life Science Award

Molecular Probes® The Handbook
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