CellCycleFlowEx Kit. PE-Cy 5 PerCP-Cy 5.5 PE-Cy 7 APC-Cy 7. Tandem dyes for your multicolor flow cytometry panel. summer 2014

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1 summer 2014 information magazine for partners EXBIO Praha, a.s. Our Mission EXBIO Praha strives to exceed the most demanding customer expectations in the field of analytical cytometry by providing a comprehensive portfolio of high quality products and services at affordable prices. New Kit for cell cycle analysis CellCycleFlowEx Kit This kit is intended for cell cycle analysis based on measuring of DN content using flow cytometry. It enables to recognize the cells in G0/G1 phase (the same amount of DN as the resting cells), New conjugates for your flow cytometry from EXBIO If you order 100 test vial of any PC-Cy 7 products, you will get 25 test vial of another PC-Cy 7 conjugate for free. Please mention discount code CQ on your purchase order. Valid until September 30, S phase (in the process of DN duplication), and G2/M phase (having double the amount of DN as the resting cells). more on page 4 more on page 3 Tandem dyes for your multicolor flow cytometry panel -Cy 5 PerCP-Cy 5.5 -Cy 7 PC-Cy 7 1

2 HOT Topics Newly dded Clones With respect to our customers needs we are continuously searching for valuable hybridomas to supplement our portfolio with high quality antibody clones, and we are also developing our own ones. Here we bring a table of clones that have been added to our portfolio since last issue. s your needs and requirements are of high importance for us, we are grateful for you feedback what other important clones you would like to see in our portfolio. Catalog No. ntigen Clone Host Isotype Reac. pplication Format Quantity C C100 CD1b SN13 IgG1 Hu FC, IP, IHC(F), 1F-761-T025 1F-761-T100 IHC(P) FITC FITC C C100 CD1c L161 IgG1 Hu FC, IP, IHC 1P-752-T025 1P-752-T C C100 CD3 zeta Phospho-Tyr 153 EM-17 IgG1 Hu, FC, WB C C100 CD41 MWReg30 Rt IgG1 FC, IP, IHC(P), 1P-763-C025 IHC(F), 1P-763-C100 ELIS, FUNC: platelet depletion C C100 CD45R R3-6B2 Rt IgG2a, Hu, FC, IP, IHC(P), C100 Fel IHC(F), ICC, low endotoxin 1F-715-C025 FUNC: FITC 1F-715-C100 modulation FITC of B cell 1P-715-C025 responses 1P-715-C C C100 PC PC C C100 CD56 LT56 IgG2a Hu FC, WB, ELIS 1P-789-T025 1P-789-T C C100 CD88 S5/1 IgG2a Hu, Bov, FC, IP, WB, IHC(P) 1P-736-C025 Rb, 1P-736-C100 Fer C C100 CD160 BY55 IgM Hu FC, IP C C100 CD266 ITEM-4 IgG2b Hu, FC, WB, IHC(F), C100 FUNC: low endotoxin 1P-755-C025 blocking 1P-755-C C C100 NCK1(an adaptor protein) EM-06 IgG1 Hu, WB, ICC C C100 GPS (a peroxisomal enzyme) GPS-03 IgG2a Hu FC, WB C C100 CLIC5a (component of actin CLIC5-02 IgG2b Hu FC, WB 1P-701-C025 1P-701-C100 complexes) C C100 LRGE1 (a glycosyltransferase) LRGE-02 IgG2b Hu FC, WB 1P-702-C025 1P-702-C C C100 DDIT4L (a stress-induced DDIT-03 IgG1 Hu FC, WB 1P-725-C025 1P-725-C100 protein) C C100 OPL1 (a transmembrane adaptor protein) OPL1-01 IgG2a Hu FC, WB 1P-699-C025 1P-699-C100 Reactivity of the monoclonal antibody EM-17 (anti -CD3 zeta phospho -Tyr153) with phosphorylated particular human CD3 zeta mutants. The Y6F mutatant lack phosphotyrosine 153 Compared with reactivity of EM-26 (anti-cd3 zeta phospho-tyr72). bbreviations Hu = human Pri = primates Por = porcine = mouse Rt = rat Bov = bovine Western blotting analysis of NCK1 in whole cell lysate of mouse lymph node lymphocytes using the antibody EM-06. The antibody was approved also in Borroto. et al., J. Immunol Feb 1;190(3): Western blotting analysis of DDIT4L expression in HEK293-DDIT4L transfectants and HEK293 cells using mouse monoclonal antibody DDIT-03. Gui = Guinea pig Can = canine Eq = equine Rb = rabbit Chk = chicken Western blotting analysis of OPL1 in OPL1-transfected HEK293 cells using mouse monoclonal antibody OPL1-01. PC Cy 7 tandem fluorochrome Near -Infra Red workhorse Most of the flow cytometers currently used in clinics, and probably every cytometer used in research, are equipped with red laser emitting at around 635 nm. Red laser offers a great opportunity to use antibody conjugates in red, far -red and near infra-red channel in your multicolour panels. The number of possible fluorescent labels usable for NIR channel is very limited. There are practically no small organic dyes that are excitable by red laser at 635 nm wavelength with high fluorescence yield and emission in NIR spectrum. This obstacle can be overcome by using an PC tandem fluorochrome which not only is excitable by red laser with high efficiency but also fluoresces in NIR part of visible spectra. The first one ever used and one of the best around is PC-Cy 7 tandem fluorochrome. Excitation properties of PC-Cy 7 are the same as for PC with absorption maxima at around 650 nm. Fluorescence maxima of PC-Cy 7 tandem fluorochrome peaks at around 780 nm making it exceptionally suitable for use with 780/60 nm NIR detector. Here, PC (llophycocyanin) a red emitting (at 660 nm) fluorescent protein acts as an energy donor when excited using 635nm red laser. fter PC absorbs energy in the form of photons it becomes excited and readily releases absorbed energy through a non -fluorescent energy transfer via electron clouds of accumulated double and triple bonds to Cy 7 fluorochrome which releases the acquired energy in the form of near -infra red fluorescence. PC-Cy 7 Example data CD3 CD4 CD8 CD19 Blue line represents Isotype control and Red line represents PC -Cy 7 antibody conjugate staining. In this case Cy 7 or so -called energy acceptor is covalently linked to PC protein to a location which is in a very close vicinity of PCs chromophores in order to achieve the highest efficiency of energy transfer. Lower energy transfer efficiency would negatively influence free PC fluorescence eventually increasing the spillover of PC-Cy 7 tandem into PC channel resulting in errors in your fixed compensations. n extreme care and a sensitive approach has been taken when we were developing PC-Cy 7 tandem fluorochrome for the sake of its high stability in order to avoid short conjugate shelf -life. Quick deterioration has been a known issue of PC-Cy 7 conjugates previously. This can be seen as an increase in free PC fluorescence also called PC spillover. fter we selected Cy 7 to be the organic dye of choice for an PC tandem we had to optimize the tandem dye in order to achieve the highest fluorescence possible, decrease the PC -Cy 7 spillover into PC channel to a reasonable level, decrease tandem non -specific binding and finally: to achieve the highest PC - Cy 7 tandem stability possible. The whole product development took a lot of time and effort, but now we can present the last red laser color you have been waiting forin our portfolio. lthough PC-Cy 7 is not considered to be a bright fluorochrome, it is perfectly suitable for staining of CD3, CD4, CD8, CD19 and other common extracellular antigens. Normallized Emission (%) Wavelength (nm) Excitation: 633 nm Emission max.: 774 nm Brightness: Recommended filter: 780/60 Tandem dye* of allophycocyanin and Cy 7 *Tandem dyes need lot-specific compensation. Catalog No. Product Quantity T4-514-T025 T4-514-T100 T4-359-T025 T4-359-T100 T4-207-T025 T4-207-T100 T4-663-T025 T4-663-T100 T4-366-T025 T4-366-T100 T4-222-T025 T4-222-T100 T4-586-T025 T4-586-T100 T4-673-T025 T4-673-T100 T4-632-T025 T4-632-T100 Mb to CD3 (UCHT1) PC-Cy 7 Mb to CD4 (MEM-241) PC-Cy 7 Mb to CD8 (MEM-31) PC-Cy 7 Mb to CD19 (4G7) PC-Cy 7 Mb to CD38 (HIT2) PC-Cy 7 Mb to CD45 (MEM-28) PC-Cy 7 Mb to CD117 (104D2) PC-Cy 7 Mb to Lambda light chains ( ) PC-Cy 7 IgG1 Isotype control PC-Cy 7 Miroslav Benko, Senior Product Development Scientist 2 3

3 New Kit : Viable cell Cell Cycle nalysis with Catalog no. Product name mount ED7069 CellCycleFlowEx Kit 200 tests CellCycleFlowEx Kit is intended for cell cycle analysis using flow cytometry. The kit is suitable for suspensions of isolated cells, such as peripheral blood leukocytes or cells from tissue culture. The test is based on measuring of DN content within the individual cells by detection of intercalated propidium iodide molecules. Cellular DN content within a proliferating cellular population differs between the particular phases of the cell cycle, and its measurement enables to recognize the cells in G0/G1 phase (the same amount of DN as the resting cells), S phase (in the process of DN duplication), and G2/M phase (having double the amount of DN as the resting cells). nalysis of the percentage of cells at different stages of the cycle according to their DN content is being used to assess the proliferative response to stimu- lation with mitogens (lymphocyte transformation tests), or to monitor the effect of cell cycle inhibitors. The tested cells are washed and resuspended in a buffer to achieve a homogeneous suspension. Then the cells are fixed and permeabilized with 70% ethanol. Subsequently the cellular DN is stained with propidium iodide, which passes through the permeabilized membrane and intercalates between the bases of nucleic acid. Propidium iodide binds to the DN stoichiometrically, which means that the amount of bound propidium iodide is proportional to the amount of DN within the cell. Labeling of DN takes place in buffer with RNse, which digests the contaminating cellular RN. fter laser excitation such cells emit fluorescence of whichintenzity is proportional to the DN content. B B Delimitation of the target cell population : PBMC stimulated with PH (5μg/ml) for 3 days B: unstimulated controls Delimitation of singlets : stimulated PBMCs B: controls Developer s Notes We have taken a lot of effort to develop a product that would provide standardized high quality results achievable with just a few pipetting steps. The task was to design a simple and robust detection method for measuring the proliferative response in suspended cells. The CellCycleFlowEx Kit staining protocol and its reagent formulations are designed: 1) To keep debris and aggregates to a lower or equal level that is achievable with other kits in the market. The assay gives the best results if the sample originates from homogeneous cell population, the most suitable are blood leukocytes and cell lines with a low level of aneuploidity. 2) To enable stopping the procedure to collect more samples or to transport them to another lab. 3) To enable also the detection of apoptotic cells. Those are revealed as the cellular events with decreased stainability (sub G1) which is caused by the leakage of fragmented DN from ethanol permeabilized cellular membranes. The other advantage of the CellCycleFlowEx Kit is that the protocol prevents excessive cell aggregation observed with many other kits in the market. CellCycleFlowEx Kit brings to users a convenient tool to analyse proliferative responses of suspended cells, which is suitable e.g. when studying effects of proliferation inhibitors (anti-cancer drugs), or effect of stimulators of the immune system (lymphocyte transformation test, LTT). Last, but not least it can be used for detection of apoptotic cells. B: Early apoptotic cell C: Death cell Phosphatidylserine (PS) nnexin V FITC Propidium Iodide (PI) Catalog no. Product name mount ED7044 poflowex FITC Kit EXB0024 nnexin V FITC EXB0027 nnexin V EXB0028 nnexin V PC EXB0023 nnexin V Dyomics 647 EXB0019 nnexin V Binding Buffer (10 ) 50 ml EXB0018 Propidium Iodide poptosis is a programmed cell death mechanism that is characterized by a variety of morphological features such as loss of membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DN. One of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. Once exposed to the extracellular environment, binding sites on PS become available for nnexin V, a kda, Ca 2+ dependent phospholipid-binding protein with a high affinity for PS. nnexin V is a sensitive probe for identifying cells that are undergoing apoptosis because PS exposure occurs early in the apoptotic process. It is typically conjugated to a fluorochrome, such as FITC (fluorescein isothiocyanate), for easy identification of apoptotic cells by flow cytometry. nnexin-v FITC identifies cells at an earlier stage of apoptosis than assays based on DN fragmentation. Since membrane translocation of the PS also occurs during necrosis, nnexin V is not an absolute marker of apoptosis. It is often used in conjunction with vital dye such as propidium iodide (PI), which binds to nucleic acids, but can only penetrate the plasma membrane when membrane integrity is breached, as occurs in the later stages of apoptosis or in necrosis. Therefore, cells that are negative for both nnexin V and the vital dye have no indications of apoptosis, as PS translocation has not occurred and the plasma membrane is still intact. Early apoptotic cells are nnexin V-positive and vital dye-negative, as PS translocation has occurred, but the plasma membrane is still intact. However, the assay does not distinguish between cells that have already undergone an apoptotic death and those that have died a necrotic death because they both stain similarly with nnexin-v FITC (positive) and PI (positive). Panel of antibodies to the antigens involved in apoptosis is available at Would you need an assistance, please do not hesitate to contact us at als@exbio.cz B Distribution of cells according to the DN content into the G0/G1, S and G2/M phases of the cell cycle : stimulated PBMCs B: controls Pavel Jinoch, Senior ssay Development Specialist poptosis was detected using poflowex FITC Kit in the Jurkat cell line after incubation with TRIL (TNF-related apoptosis inducing ligand). Green line: unstimulated unstained cells, red line: unstimulated stained cells, blue line: TRIL-stimulated stained cells. Early apoptotic cells are annexin V positive and propidium iodide negative. 4 5

4 More from EXBIO EXBIO Posters Our Custom Services New poster available For more than twenty years EXBIO has been a primary manufacturer of high quality monoclonal antibodies. Our experienced staff is pleased to offer the resources available from EXBIO including our quality certified facilities, time saving expertise and proprietary techniques to researchers world wide through our selection of custom services. Monoclonal ntibody Production Our latest poster Lymphocyte Immunophenotyping describes our CE IVD reagents for immunophenotyping of human blood, including lysing solutions. M. Stránský michal@sky.cz zakázka: EXBIO poster papír: km ca 150g Cyan Magenta Yellow Lymphocyte Immunophenotyping 2 Color Combinations (CE-IVD) Select one of the colors we have in our portfolio (including tandem dyes) and let EXBIO conjugate the protein or antibody of your choice using successfull conjugation process. Conjugation with biotin is also available. Diagnostics Red Blood Cell Lysis 6 Red Laser Pacific Blue lexa Fluor 488 PC Pacific Orange FITC lexa Fluor 647 lexa Fluor 700 -DyLight 594 PC-Cy 7 -Cy 5 FITC / CD4 / CD8 FITC / CD3 / CD19 FITC / CD3 / CD16+CD56 FITC / CD3 / HL-DR FITC / CD45 / CD14 FITC / Negative Control FITC / CD4 / CD8 / CD3 FITC / / -Cy 5 CD4 / CD8 / CD3 FITC / / PerCP CD3 / CD16+CD56 / CD45 FITC / / PerCP CD3 / CD19 / CD45 FITC / / PerCP CD3 / CD4 / CD45 FITC / / PerCP CD3 / CD8 / CD45 FITC / / PerCP CD3 / CD8 / CD45 / CD4 FITC / / PerCP / PC CD3 / CD16+CD56 / CD45 / CD19 FITC / / PerCP / PC CD3 / CD8 / CD45 / CD4 -Cy 5 / / FITC / -Cy 7 CD3 / CD16+CD56 / CD45 / CD19 -Cy 5 / / FITC / -Cy 7 ntibodies Multicolor Reagents CD3 / CD16+CD56 / CD45 / CD4 / CD19 / CD8 FITC / / PerCP-Cy 5.5 / -Cy 7 / PC / PC- Cy 7 EXCELLYSE I 30 ml ( tests) 100 ml ( tests) Features: Red blood cell lysis and white blood cell fixation Suitable for both lyse/wash and lyse/no wash lysing procedure Excellent separation of lymphocytes in SSC vs. FSC dot plot 2-step lyse/no wash procedure B cell EXCELLYSE Easy 30 ml ( tests) 100 ml ( tests) Features: Red blood cell lysis and white blood cell fixation Suitable for both lyse/wash and lyse/no wash lysing procedure Quick and easy protocol 1-step lyse/no wash procedure Suitable when lymphocyte gating through CD45 or other marker is performed. EXBIO Praha, a.s. Nad Safinou II Vestec Czech Republic tel.: fax: orders@exbio.cz Lysing Solutions (CE-IVD) ssays and Kits Suitable even when lymphocyte gating through CD45 or other marker is not performed. Standardized multicolor flow cytometry panels have become one of the basic tools in hematooncology and immunology. We are in close contact with our customers to provide them with those fluorescent conjugates they really need for their panels. lso fluorescence microscopy has its particular needs regarding. Summarizing all feedbacks we are continuously updating our range of fluorescent dyes conjugated with primary antibodies. lexa Fluor, Pacific Blue and Pacific Orange are registered trademarks of Life Technologies Corporation. DyLight is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries. Cy and CyDye are registered trademarks of GE Healthcare. FITC / CD3 / CD8 6 Color Combination (CE-IVD) Colors of EXBIO Overview Blue Laser CD3 / CD4 4 Color Combinations (CE-IVD) Custom Services Violet Laser 3 Color Combinations (CE-IVD) Production of Rabbit Polyclonal ntibody ccording to your demands we will deliver you rabbit antiserum or polyclonal antibody. Contact us at als@exbio.cz Write us to orders@exbio.cz black Custom conjugation Reliable and cost effective, EXBIO is ready to maintain your hybridoma and produce on demand your mouse, rat, or hamster monoclonal antibodies in bulk quantities. You supply the hybridoma and we will produce the antibodies following a proven process that yealds the highest quality results. Would you like to have one of our posters? CD4+T cell NK cell CD8+T cell Cy and CyDye are registered trademarks of GE Healthcare. CE-IVD reagents conform to the European In Vitro Diagnostic Medical Device Directive 98/79/EC. Our older posters Examination of allergies and defects of phagocytosis has been described in these two posters: Summary of spectral characteristics of thirty common being used in flow cytometry you can find in the poster Flower Field Guide : PerCP PerCP-Cy 5.5 -Cy 7 7

5 Where you can meet us: CC 2014 July Chicago, US ESCC 2014 September Lisabon, Portugal 24 th nnual Conference of the German Society for Flow Cytometry October Dresden, Germany ESID Satellite on Flow Cytometry November 1 2 Prague, Czech Republic pplication Form We re Grateful for Papers More precisely: We are grateful for your articles We appreciate you use EXBIO reagents and we would like to thank you with the product credit for your next order. Product credit (according to invoice currency) 250 USD 200 EUR 5000 CZK What needs to be done to get credit for publication? How does it work? Fill the We are grateful for papers pplication Form (available on our web site: ) filled form and the article to technical@exbio.cz Our sales team will contact you with a special code for your next order Publication Information rticle Title Journal Title Personal Information Name Institution Department Shipping ddress We are grateful for papers pplication Form Terms and Conditions: EXBIO must be cited in the article only in the form: EXBIO ny author can submit the article. We give you 1 product credit per article only. Journal article must be peer reviewed. Product credit is valid on catalog items only, not for custom service. Product credit expires in 12 months from date of issue. Offer is valid worldwide. Void where prohibited. It is the submitter s responsibility to adhere to their institution s policies and guidelines. Joint nnual Meeting of French Society for Immunology and the French Cytometry ssociation November 4 6 Lille, France Medica 2014 November Düsseldorf, Germany EuroBT 2014 December München, Germany bbreviations: PC... allophycocyanin FITC... fluorescein isothiocyanate... R-phycoerythrin PerCP... peridinin chlorophyl a protein RUO... research use only CE-IVD... in conformity with the European... In Vitro Diagnostic Medical... Device Directive 98/79/EC. Contacts of your article to technical@e xbio.cz Country Phone ll fields must be filled. Please send filled form along with a PDF copy of your article to technical@e xbio.cz EXBIO Praha, a.s. Nad Safinou II Vestec u Prahy tel fax orders@exbio.cz technical@exbio.cz lexa Fluor, Pacific Blue and Pacific Orange are trademarks of Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corporation); DyLight is a trademark of Thermo Fisher Scientific; Cy is a trademark of GE Healthcare Limited. 8