The most complete fluorescent labeling and detection reference available, The Molecular Probes HandbookA Guide to Fluorescent Probes and Labeling Technologies contains over 3,000 technology solutions representing a wide range of ... Instead of PI, 4',6 … Found inside – Page 86Follow the manufacturers protocol for annexin - V staining ( Figure 5a ) ... at 1 x 10/100 ul in 1.5 ml microfuge tubes and stained with annexin V - FITC and ... It is used to test a protocol to make sure it works. Found inside – Page 93Yeast displaying wild-type Vβ TCR were stained with (A) KJ16- FITC, ... while the unshaded peak represents the population for positive FITC staining. Found inside – Page 49In a recent study, it has been demonstrated that FITC-PSA/PI-staining protocol can detect marked deterioration in both the PMI and AMI in frozen-thawed bull ... Found inside – Page 114(See Comments under Protocol 2.) ... Furthermore, not all microorganisms stain equally well, if at all, with FITC due to hydrophilic surface structures that ... Found insideThis book stems from the editors'experiences managing flow cytometry/cell sorting core facilities for the emerging researchers, in particular in developmental, cellular, and molecular biology. This volume presents the latest collection of immunophenotypic techniques and applications used in research and clinical settings. Found inside – Page 268Check the FITC staining with the 488-nm filter. Note, the CY5 647-nm wavelength is not visible with light from the mercury UV lamp and can be viewed only ... Found inside – Page 837Figure 3A ows a plot of red versus green fluorescence for FITC - stained pocotyl protoplasts . ... obtained from the fusion mixThe development of flow cytometric protocols for sorting plant protoplasts could greatly increase the usefulness and ... Learn about our antibody labeling kits and their advantages. Annexin V-FITC Apoptosis Staining / Detection Kit ab14085 is used in a 10 min, one-step staining procedure to detect apoptosis by staining phosphatidylserine molecules which have translocated to the outside of the cell membrane. 20. Found inside – Page 517The first protocol is used for cell surface staining. ... and indirectly stained ; or multiple cell surface proteins using fluorescein isothiocyanate (FITC) ... The iFluor dyes are brighter and more photostable than traditional dyes such as FITC and rhodamine and provide similar performance to Alexa Fluor ® dyes. 2009) Fig. Background Immunofluorescence is a technique that allows the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). Written by known specialists in the field, this is a comprehensive and timely overview of a central and expanding topic. Bioconjugate Techniques, Third Edition, is the essential guide to the modification and cross linking of biomolecules for use in research, diagnostics, and therapeutics. As in the cell surface staining protocol, optimize the concentration of each antibody to be used in staining. Found inside – Page 186The optical density of the FITC-conjugated antibodies is measured at 495 nm ... used in a direct immunof- luorescent staining protocol for flow cytometry. Annexin V Staining Protocol General Annexin V Staining Procedure Solutions 10X Binding Buffer (cat. If samples are to be fixed post-staining, there are specific conditions required to achieve transient retention of signal. This volume provides an overview of commonly used methods and protocols for cell fitness indicators. Found inside – Page 262See fluorescent in situ hybridization FITC-ConA staining. ... DIOC6(3), Rh123, CMXRos and CMreactive H2XRos oxygen staining species protocol detection for, ... Found inside – Page 121The staining protocol absolutely must be carried out in Annexin V-binding ... cells stained only with Annexin V-FITC (no PI added) and cells stained ... Found inside – Page 16Annexin V-FITC bound cells show green staining in the plasma membrane and cells that have lost membrane integrity will show red staining (PI) throughout the ... Found inside – Page 194Fluorescein isothiocyanate (FITC, 495/519 nm ex/em), a derivative of ... Lectins are combined with FITC to stain spermatozoa or other target cells. It can also be used to conjugate other proteins or peptides. In fixed cells, actin structures can be visualized by fluorescent phalloidins (2, see protocol below), actin antibodies (1, see protocol below), or electron microscopy (3).Correctly designed fluorescent phalloidins only binding to the native quaternary structure of F-actin and therefore have a low background. Found inside – Page 33(A) High magnification picture of SSEA-4 cell surface antigen staining localized with FITC labelled secondary antibody. (B) DAPI counter stain of A. (C) Two ... Found inside – Page 55If the only reagents available to stain a pair of markers on a given cell ... a determinant detected by a FITC-coupled reagent and the other does not. You have to find a tissue that has known positive antigen of interest using as a positive control tissue. no. Found inside – Page 264Mock transfected or MDM2 expression plasmid transfected cells were fixed and stained with PE-labeled MDM2 antibody, FITC-labeled anti-BrdU antibody, ... Annexin V Staining Protocol General Annexin V Staining Procedure Solutions 10X Binding Buffer (cat. Propidium Iodide (PI, cat. Found insideFlow cytometry is a technique widely used in biological research and in diagnostic medicine. Flow cytometers are found in most biological research institutions and most clinical laboratories in larger hospitals. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE). A multi-color immunofluorescent experiment has one FMO control for each fluorochrome. no. Jurkat cells were treated with 10 μM EdU for one hour and stained with Invitrogen Alexa Fluor 488 picolyl azide, according to the protocol for the Invitrogen Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit, followed by staining with Invitrogen FxCycle Violet Stain. Another important considerationof a fixation protocol is the buffer selection. One consideration when performing multicolor fluorescence studies is the possibility of spectral overlap between fluorophores. 2 Principle Annexin V staining to detect apoptotic cells can only be done on live cells and tissue. Application Note we present a staining protocol using fluorescein diacetate (FDA) and propidium iodide (PI), which stain viable cells and dead cells, respectively. Because the fluorophores used in flow cytometry emit photons of multiple energies and wavelengths, a mathematical method called compensation was developed to address the measurement of the photons of one fluorophore in multiple detectors. Found insideThis volume describes easy to follow methods to guide both the novice and more experienced researcher seeking to use new techniques for the culture of cancer cells. Found inside – Page 309In this protocol the amount of the greenFITC and the red-PY fluorescence will ... cytoplasm will require lower FITC and PY dye concentrations for staining ... - Phalloidin FITC. This is also something that we often want to do in flow cytometry experiments. One consideration when performing multicolor fluorescence studies is the possibility of spectral overlap between fluorophores. This volume of the acclaimed Methods in Cell Biology series provides specific examples of applications of confocal microscopy to cell biological problems. Surface staining of the cells was performed by incubating with FITC-, and PerCP-conjugated monoclonal antibodies specific to CD3, CD4, and CD19 for 30 min on ice. 556454): 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2. This is also something that we often want to do in flow cytometry experiments. Recommended for use in parallel with Annexin V-FITC (cat. Found inside – Page 52Resuspend the cell pellet with 50 l staining solution and add donkey APC- ... the direct staining method with the FITC-conjugated anti-active caspase-3 ... Found inside – Page 409Perform immunofluorescence staining if subset analysis is desired. ... When staining by TUNEL alone, use the bivanant histogram FITC and 90° light scatter ... If samples are to be fixed post-staining, there are specific conditions required to achieve transient retention of signal. no. Found inside – Page 30The rhodamine-labeled oligonucleotide probe in conjunction with an indirect IF protocol based on specific MAb was applied. This MAb with a FITC-labeled ... RPE and FITC, Takizawa et al. After doing my regular staining with primary and secundary-tritc cojugated antibody, on methanol fixed cells, I saw a typical staining of my protein (nuclear and cytoplasmic). Found inside – Page 329This staining protocol evolved from testing a range of dye concentrations ... FLUORESCENCE Chromatin can be simultaneously stained with PI and FITC . anti-CD3-FITC, anti-CD4-Biotin, and anti-CD8-APC) and incubate on ice for 15-20 minutes in the dark. It is used to test a protocol to make sure it works. You have to find a tissue that has known positive antigen of interest using as a positive control tissue. Target antigen detection methods. Cell-Surface Staining with Antibody: Add appropriately conjugated fluorescent, biotinylated, or purified primary antibodies at predetermined optimum concentrations (e.g. 556420, 556419) or Annexin V-Biotin (cat. FITC Conjugation Kit / FITC Labeling Kit (Fast) (ab188285) uses a simple and quick process for FITC / Fluorescein labeling / conjugation of antibodies. Found inside – Page 420MT1-MMP trans- 1 cells stained with normal sheep serum IgG show tracts of film clearance 3 staining on the FITC channel (10) indicating that the FITC ... no. Found inside – Page 572.5 Phagocytosis/Autophagy Assay THP-1 cells were double stained using a typical protocol for phagocytosis. Cells were incubated for 2 h with FITC-labeled ... ... Anti-EGFR antibody covalently labeled with FITC and incubated with unpermeabilized SK-BR-3 cells counter stained with Hoechst 33342. FMO controls are used to determine the cut-off point between background fluorescence and positive populations in multi-color immunofluorescent experiments. With wide varieties of antibodies, fluorophores, and instruments available for multicolor flow cytometry, optimizing your multicolor experiments can be confusing and frustrating. The direct method is a one-step staining method and involves a labeled antibody (e.g. Analysis is by flow cytometry or fluorescence microscopy. ... FITC is the workhorse of most flow cytometry labs. Add properly diluted primary antibody to cover the sample; Place the slide into a wet box and incubate at 37℃ for 1-2 hours To conjugate an antibody to FITC / Fluorescein using this kit: The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for cell viability staining using propidium iodide. Use our Multicolor Staining Guide to assist you in developing and optimizing your flow cytometry experiments. Instead of PI, 4',6 … The above is an example protocol for PS exposure detection using annexin V, based on the protocol provided in Annexin V-FITC Apoptosis Detection Kit (ab14085) . no. Any product specific protocol supercedes these general recommendations. The staining protocol is applicable to adherent cells, single cells embedded in extracellular matrix and 3D cell clusters, for example multicellular spheroids. Then I put on -20 the slides and after one week I took back the slides, and stained another protein with a FITC-conjugated secundary antibody. Found inside – Page 175If the protocol calls for several different antibody specificities coupled to a given ... Data on experimental groups stained with anti-Thy1.2 FITC (high ... The following protocol has been developed and optimized by R&D Systems Flow Cytometry Laboratory for cell viability staining using propidium iodide. Found insideFlow cytometry - Select Topics is a collection of chapters that illustrate the constantly evolving application of flow cytometry to diverse areas of research or clinical investigations. Practical and adaptable, Mammalian Cell Viability: Methods and Protocols serves as a self-contained laboratory manual useful to both experienced researchers and those new to this incredibly important and influential field. Found inside – Page 8268For isolation of IgM and IgM- B cells , cells were first stained with an allophycocyanin - conjugated anti - human IgM ... V - FITC and propidium iodide staining were performed according to the manufacturer's protocol ( BenderMedSystems ) . no. Introduction to immunochemistry for molecular biologists and other nonspecialists. Spiral. This thoroughly revised and updated edition of a widely used practical guide to flow cytometry describes in step-by-step detail an array of time proven and cutting-edge techniques much needed in today's advanced laboratories. no. Then I put on -20 the slides and after one week I took back the slides, and stained another protein with a FITC-conjugated secundary antibody. Found insideA Springer Lab Manual Review of the First Edition: "This is a most useful volume which will be a welcome addition for personal use and also for laboratories in a wide range of disciplines. Highly recommended. Immunofluorescence assay. If the positive control tissue showed negative staining, the protocol or procedure must have problem and needs to be tested until a good positive staining is resulted. 556463). Cell-Surface Staining with Antibody: Add appropriately conjugated fluorescent, biotinylated, or purified primary antibodies at predetermined optimum concentrations (e.g. (A) A diagram illustrating the procedure involved in IFA. Dilute to 1X prior to use. Actin Staining in Fixed Cells. Isotype controls. Fixation is routinely used in histology and cytology Labs the world over as a way of keeping cells in stasis at a particular point to ensure that, by the time they are examined, they have not deteriorated. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE). After cells grown on cover glass are fixed and permeabilized, a primary antibody is added to detect specific antigen, and the secondary antibody conjugated to fluorescence tag (eg, FITC) is sequentially added to bind to the primary antibody. Fluorescence-activated cell sorting (FACS) is a laser-based, biophysical technology that allows simultaneous multiparametric analysis. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in contrast to indirect approaches. Because the fluorophores used in flow cytometry emit photons of multiple energies and wavelengths, a mathematical method called compensation was developed to address the measurement of the photons of one fluorophore in multiple detectors. Found inside – Page iiIn The Protein Protocols Handbook, I have attempted to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benehtop manual and guide both for those who are new to the protein chemistry ... We recommend our Phalloidin-iFluor dye conjugates, these are available as individual reagents and in a complete F-actin staining kit format. Fluorescence-activated cell sorting (FACS) is a laser-based, biophysical technology that allows simultaneous multiparametric analysis. If the positive control tissue showed negative staining, the protocol or procedure must have problem and needs to be tested until a good positive staining is resulted. Found insideA cell cycle profile was obtained by staining cells with PI (for DNA) and FITC (for protein content) (see Protocol 4.4). This is the method of choice in the ... Found inside – Page 101... first stained with Hst and PY, followed by staining with The following staining protocol can be used when a FITC -conjugated face marker is needed (Fig. Any product specific protocol supercedes these general recommendations. Figure 4.2. For the analysis of dying cells, fluorescently labeled Annexin V (Annexin V(FITC)) and propidium iodide (PI) are the most commonly used reagents. Jurkat cells were treated with 10 μM EdU for one hour and stained with Invitrogen Alexa Fluor 488 picolyl azide, according to the protocol for the Invitrogen Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit, followed by staining with Invitrogen FxCycle Violet Stain. This first edition volume demystifies the complex topic of flow cytometry by providing detailed explanations and nearly 120 figures to help novice flow cytometry users learn and understand the bedrock principles necessary to perform basic ... FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. Fixation is routinely used in histology and cytology Labs the world over as a way of keeping cells in stasis at a particular point to ensure that, by the time they are examined, they have not deteriorated. Ensure the observed staining is due to specific binding rather than an artifact Reveal other non-specific binding of the antibody or fluorophores to cellular components (e.g. A Fluorescence Minus One (FMO) control is a tube of cells stained with all fluorochromes used in the experiment except one. This detailed volume provides a toolbox for designing constructs, tackling expression and solubility issues, handling membrane proteins and protein complexes, and exploring innovative engineering of E. coli. Affix the sample on glass slide (To ensure the validity of fluorescence staining, positive, negative and sample autofluorescence controls should be carried out to confirm there is no non-specific binding.) Found inside – Page 303Stain cells with FITC and PE, following the two-color staining protocol. In addition, make single-stained control samples for both FITC and PE, ... Add each primary antibody to each sample, making sure to stain controls as appropriate (isotype, compensation, FMO, etc.) Propidium Iodide (PI, cat. Found inside – Page 19In the three-step indirect staining protocol, cells were first incubated with FITC-NTXamine, followed by biotin-conjugated anti-fluorescein IgG, ... For the analysis of dying cells, fluorescently labeled Annexin V (Annexin V(FITC)) and propidium iodide (PI) are the most commonly used reagents. Found inside – Page 111... the labelling with the goat-anti-rabbit FITC conjugate from the staining protocol. Also test the non-specific binding of the goat-anti-rabbit-FITC. Since the publication of the frst edition, the application of immuno- tochemical techniques in the clinical laboratory has continued to rise and this third edition provides methods that are applicable to basic research as well as to the ... Found inside – Page 181In the current protocol, the Cy3 and FITC staining are described. However, there are many other possibilities to use other direct labels (e.g., like Cy5, ... 556454): 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane). 556463). 1993, Hulspas et al. After doing my regular staining with primary and secundary-tritc cojugated antibody, on methanol fixed cells, I saw a typical staining of my protein (nuclear and cytoplasmic). anti-CD3-FITC, anti-CD4-Biotin, and anti-CD8-APC) and incubate on ice for 15-20 minutes in the dark. Background Immunofluorescence is a technique that allows the visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). Dilute to 1X prior to use. no. Protocol. 556420, 556419) or Annexin V-Biotin (cat. Recommended for use in parallel with Annexin V-FITC (cat. Found inside – Page 433FITC IgGl + PerCP IgGl+ PE conj. rat IgGl FITC IgGl + PerCP IgGl + ... be required for one staining protocol and freeze this volume in bullet tubes kept at ... Annexin V staining to detect apoptotic cells can only be done on live cells and tissue. 10X Binding Buffer ( cat e.g., like Cy5, flow cytometry labs determine cut-off. Institutions and most clinical laboratories in larger hospitals V-Biotin ( cat to assist you developing. Or peptides adherent cells, single cells embedded in extracellular matrix and 3D cell clusters, for example multicellular.. With FITC and incubated with unpermeabilized SK-BR-3 cells counter stained with Hoechst 33342 ;! Optimize the concentration of each antibody to each sample, making sure to stain controls as appropriate ( isotype compensation..., anti-CD4-Biotin, and anti-CD8-APC ) and incubate on ice for 15-20 minutes the. 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And clinical settings Add appropriately conjugated fluorescent, biotinylated, or purified primary antibodies predetermined! Thp-1 cells were incubated for 2 h with FITC-labeled... found inside – Page 433FITC +...
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