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• Immunofluorescence

Protocols

• Flow Cytometry Analysis (Springer Lab, Harvard University)
  Flow cytometry employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology can provide rapid, quantitative, multiparameter analyses on single living (or dead) cells based on the measurement of visible and fluorescent light emission. Flow cytometry is a widely used method for characterizing and separating individual cells. This basic protocol focuses on: measure fluorescence intensity produced by fluorescent-labled antibodies and ligands that bind specific cell-associated molecules.
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• Advanced Methodologies in Flow Cytometry (Cytomety Lab, Purdue Univ)
  A large collection of methodological paper on cytometry
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• Annexin V Staining Protocol (BD PharMingen)
  A general protocol for staining cell for cytometry analysis.
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• BrdU Incorporation (Stewart Lab, Washington University at St. Louis)
  Label cells for flow cytometry assay
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• Direct Immunofluorescence Staining (U of Florida Flow Cytometry Core Laboratory)
  This is the method for direct immunofluorescence labeling; that is, the antibodies have the fluorescent dye attached. Direct labeling is simpler and quicker than indirect labeling.
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• FCM of leukocytes in whole blood (David Chambers)
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• Fixation and DNA Staining for Cell Cycle Analysis (U of Florida Flow Cytometry Core Laboratory)
  This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells.
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• Fixation of Immunofluorescence-Labeled Samples (U of Florida Flow Cytometry Core Laboratory)
  This fixation method is good for cells labelled by fluorochrome-conjugated antibodies to membrane antigens.
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• Flow Cytometric Analysis of Cell Cycle (Hannun/Obeid Lab)
  Protocol for fixing and staining
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• Flow Cytometry Assay to measure Internalization of GPCR's (Von Zastrow lab)
  Internalization of receptor is measured as the reduction of receptors on the plasma membrane surface. Surface receptors are labeled with fluoresent antibodies which is in turn measured using a flow cytometer. The antibodies recognize an epitope engineered onto the N-terminus of the GPCR.
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• Flow Cytometry: Indirect Immunofluorescence Staining of Human Platelets (BD Pharmingen)
  Protocol for general use.
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• Fluorescein Labeling (David Chambers)
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• Immunodetection of cyclin D1 and D2/D3 using 488/630 nm ( )
  dual laser flow cytometry(Cytomety Lab, Purdue Univ)
  This protocol is for use with the D cyclins and employs 488 nm argon laser excitation of propidium iodide and 630 nm NeNe or diode laser excitation of the fluorochrome Cy5 to detect cell cycle-specific cyclin D expression
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• Immunofluorescence Staining and Flow Cytometry of Intracellular Cytokines (eBioscience)
  A modification of the basic immunofluorescence staining and flow cytometric analysis protocol can be used for the simultaneous analysis of surface molecules and intracellular cytokines at single-cell level. In this protocol, cells are first activated in vitro, stained for surface antigens, as in the regular staining protocol, then fixed and permeabilized to allow for anti-cytokine antibodies to stain intracellularly.
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• Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method (BD PharMingen)
  Standard protocol
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• Immunofluorescent Staining of Mouse and Rat Leukocytes (BD PharMingen)
  Standard protocol including detailed procedures and tips
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• Indirect Immunofluorescence Staining (U of Florida Flow Cytometry Core Laboratory)
  This is the method for indirect immunofluorescence labeling; that is, the antibodies do not have the fluorescent dye attached.
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• Paraformaldehyde preparation ( )
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• Phenotype-specific immunodetection of cyclins using 488/630 nm dual laser flow cytometry (Cytomety Lab, Purdue Univ)
  This protocol is for use with the D and E cyclins and employs 488 nm argon laser excitation of propidium iodide and a FITC-conjugated phenotypic label, and 630 nm NeNe or diode laser excitation of the fluorochrome Cy5 to detect cell cycle-specific cyclin D expression.
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• Phycoerythrin Labeling (David Chambers)
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• Protein (Antibody) Fluorescent Labeling (Mario Roederer)
  Gives detailed protocol for conjugation of proteins (principally monoclonal antibodies) to fluorescent dyes. The conjugations fall into four basic protocols: Type 1 (used for FITC, Cy5, and the initial preparation of the Cy5 and Cy7 tandem dyes); Type 2 (used for Biotin and Cascade Blue); Type 3 (used for conjugation of PE, APC, TR-BSA, and their derivatives); and Type 4 (used for the initial preparation of non-immunoglobulin proteins like Annexin V).
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• Simultaneous analysis of DNA content and surface immunophenotype using gentle ethanol fixation (Cytomety Lab, Purdue Univ)
  This procedure utilizes gentle ethanol treatment techniques, which permeablize cells with minimal loss of surface antigen expression and antibody-antigen association.
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• Staining Procedure for Flow Cytometric Detection of Human Cyclins (BD PharMingen)
  This is a standard protocol used at Pharmingen for Quality Control testing of the anti-cyclin antibodies by flow cytometry. It is a good starting point. However, investigators may need to optimize protocols for their own experimental system. It is particularly useful to refer to the published literature regarding protocols typically used for a given type of protein.
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• Standard Flow Cytometry (FACS) Protocol (Jackson Lab)
  FACS Analysis Using Peripheral Blood Cells
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• Yeast Cell Cycle Analysis by Flow Cytometry (Susan Forsburg)
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