Evaluation of PBXL cyanobacteria antennae complexes and CryptoFluor phycobiliproteins for flow cytometry.

PBXL-1 and PBXL-3.  These novel fluorochromes were developed by Martek Biosciences Corporation and can be obtained from them or from KPL or Intergen. They are fluorescent stabilized photosystem II antennae complexes derived from cyanobacteria and are composed of multimeric, branched chains of either phycoerythrin, phycocyanin and allophycocyanin molecules with an APC core.   The branched structure of these complexes allows extensive energy transfer to occur toward the center of the complex, resulting in a potential fluorescence level well above individual molecules of PE or APC.  These complexes can be conjugated to avidin, making them potentially useful for the detection of poorly expressed antigens.   Their extremely high molecular weight can cause steric hinderance, however, making their usefulness limited to particular labeling systems.  PBXL-1 can be excited by a 488 nm argon-ion laser and PBXL-3 by a helium neon or red diode laser; both are detected using an APC or Cy5 narrow bandpass filter (660 to 680 nm).

PBXL-1             PBXL-3             Comparison of APC, CF-2 and PBXL-3 excitation spectra.

(Below).  Schematic representation of PBXL structure (in this case PBXL-1). 

(Below).  PBXL-1 detection of human CD30 on HH T lymphoma cells.  All samples were labeled with biotin-conjugated anti-human CD30 antibody followed by avidin conjugated of the indicated fluorochromes at the indicated amount.  Cells were then analyzed on a FACSVantage using either an argon-ion laser emitting at 488 nm (for PBXL-1, left panel)or a krypton-ion  laser emitting at 530 nm (for PBXL-1, right panel).

(Below).  Comparison of FITC, Cy5 and PBXL-3 detection of human CD30 on Jurkat T cells.  All samples were labeled with biotin-conjugated anti-human CD30 antibody followed by avidin conjugated of the indicated fluorochromes at 2 µg/sample.  Cells were then analyzed on a FACSVantage using either an argon-ion laser emitting at 488 nm (for FITC) or a HeNe laser emitting at 632 nm (Cy5 and PBXL-3) for fluorochrome excitation.  Cy5 and PBXL-3 were detected through a 675 + 20 nm narrow bandpass filter.  Values on each histogram indicate the fold-increase in mean fluorescence intensity of the antibody-labeled cells over background.

References.

  Telford, W.G., Moss, M.M., Morseman, J.P. and Allnutt, F.C.T. (2001) Cyanobacterial stabilized phycobilisomes as fluorochromes for extracellular antigen detection by flow cytometry. Journal of Immunological Methods 254, 13-30.

CryptoFluor-1.  The CryptoFluor phycobiliproteins are derived from cryptomonad algae and possess a molecular weight considerably lower than phycoerythrin or allophycocyanin, reducing the potential for steric hinderance during extracellular immunophenotyping and making it potentially applicable for intracellular labeling.  CryptoFluor-1 has excitation and emission characteristics similar to APC, allowing it to be excited by a HeNe laser 632 nm) and detected through a 660 nm narrow bandpass filter.   At roughly molar equivalent concentrations, CryptoFluor-1 showed brightness somewhat lower than APC in our test systems.

CryptoFluor-1

(Below).  Mouse EL4 cells immunolabeled with biotinylated antibodies against Fas (clone Jo2), CD30 (clone mCD30.1) or anti-Thy1.2.   The cells were secondary labeled with  CryptoFluor-1-streptavidin at 16 ug/sample.  The cells were then analyzed on a FACSVantage SE using a helium-neon laser for excitation (632 nm) and a 660/20 nm narrow bandpass filter for emission detection.

(Below).   The CryptoFluor dyes are also being assessed for intracellular labeling.  Rat UMR-106 osteosarcoma cells were incubated on glass tissue culture slides to near-confluency and fixed with 70% EtOH.  The cells were subsequently labeled with biotin-conjugated anti-PCNA and secondary labeled with either Cy5-avidin or CryptoFluor-1-streptavidin.  The cells were then counterstained with propidium iodide (PI) at 50 ug/ml and analyzed on our Compucyte laser scanning cytometer using an argon-ion 488 nm laser to excite PI and a HeNe 632 nm laser to excite Cy5 or CryptoFluor-1.  PI fluorescence was used as the contouring parameter.

CryptoFluor-2.   CryptoFluor-2 also has excitation and emission characteristics similar to APC, allowing it to be excited by a HeNe laser 632 nm) and detected through a 660 nm narrow bandpass filter.   At roughly molar equivalent concentrations, CryptoFluor-2 showed comparable brightness to APC in our test systems.

CryptoFluor-2               Comparison of APC, CF-2 and PBXL-3 excitation spectra.

(Below).  Mouse EL4 cells immunolabeled with biotinylated antibodies against Fas (clone Jo2) or CD30 (clone mCD30.1) and human HH lymphoma cells were labeled with biotinylated anti-TNFR2.  The cells were then secondary labeled with either APC-avidin at 2 ug/sample (100 ul volume) or CryptoFluor-2-streptavidin at concentrations ranging from 16 to 2 ug/ml/sample.  The cells were then analyzed on a FACSVantage SE using a helium-neon laser for excitation (632 nm) and a 660/20 nm narrow bandpass filter for emission detection.

(Below).  To asses the usefulness of CryptoFluor-2 for intracellular labeling, mouse EL4 cells were fixed with 70% EtOH and labeled with biotin-anti-PCNA antibodies.  The cells were then secondary labeled with either Cy5- or CryptoFluor-2-streptavidin.  CryptoFluor-2 fluorescence was found to be comparable to Cy5.

CryptoFluor-4.  This  phycobiliprotein has emission characteristics similar to PE and Cy3 and is brighter than CryptoFluor-3.   It is also optimally stimulated with the 530 nm line of a krypton-ion laser and is detected through a 575 or 585 nm narrow bandpass filter.

CryptoFluor-4

(Below).  Mouse EL4 cells immunolabeled with biotinylated antibodies against Fas (clone Jo2), CD30 (clone mCD30.1) or anti-Thy1.2.  The cells were then secondary labeled with CryptoFluor-4 at the indicated amount and analyzed using the indicated excitation wavelengths and emission filters. 

(Below).  To asses the usefulness of CryptoFluor-4 for intracellular labeling, mouse EL4 cells were fixed with 70% EtOH and labeled with biotin-anti-PCNA antibodies.  The cells were then secondary labeled with either Cy3- or CryptoFluor-4-streptavidin.  CryptoFluor-4 fluorescence was found to be comparable or superior to Cy3.

CryptoFluor-5.  This  phycobiliprotein has emission characteristics similar to lissamine rhodamine and Alexa Fluor 568, with comparable brightness.   It is also optimally stimulated with the 568 nm line of a krypton-ion laser.

CryptoFluor-5.         Comparison of lissamine rhodamine, Alexa Fluor 568 and CryptoFluor-5 excitation spectra.

(Below).  Mouse EL4 cells immunolabeled with biotinylated antibodies against CD95 (clone Jo2), CD30 (clone mCD30.1) or CD90.  The cells were then secondary labeled with CryptoFluor-4 at the indicated amount and analyzed using the indicated excitation wavelengths and emission filters. 

References.

  Telford WG, Moss MW, Morseman JP, FCT Allnutt.  Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry.  Cytometry 2001;44,16-23.

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