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In Vitro Class Switching Staining for Flow Cytometry Protocol - http://www.natureprotocols.com/2007/01/22/in_vitro_class_switching_stain.php

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for in vitro class switching staining for flow cytometry. - [Read In Vitro Class Switching Staining for Flow Cytometry Protocol]

Indirect Immunofluorescence Labeling Protocol - http://www.biotech.iastate.edu/facilities/CELLHYB/Indirect.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

An Indirect Immunofluorescence Labeling Protocol for flow cytometry. Iowa State University, Flow Cytometry Facility - [Read Indirect Immunofluorescence Labeling Protocol]

Intracellular Staining for Flow Cytometric Analysis Protocols - http://www.ebioscience.com/ebioscience/appls/FCI.htm?

Category: Signalling Signal Transduction Protocols: Cytokine Protocols

Intracellular Cytokine Staining Protocol. Analysis of surface molecules and intracellular cytokines at the single-cell level using FACS or flow cytometry. - [Read Intracellular Staining for Flow Cytometric Analysis Protocols]

Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66368A929753C320BDC7C58DA3FDFD&objectid=66749F0EB1D3C751C86C30427F3C7AB0

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

A flow cytometry technique is presented, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules - [Read Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol]

Live/Dead Assay for Cell Viability Protoco - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000023.pdf?pid=PP00000023

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Assay measures cell viability. It is a two-color fluorescence assay that simultaneously determines Live cell number and Dead cell number. This protocol is designed for use with the GEMINI XS Microplate Spectrofluorometer, a multi-well plate scanner with dual excitation/emission capabilities, but the assay is also adaptable for flow cytometry and fluorescence microscopy. Includes: Cell Culture; Preparation for the Assay; Live/Dead Assay; Reading the Plate; Data Analysis; Alternative protocol. - [Read Live/Dead Assay for Cell Viability Protoco]

Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4436

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Measurement of Intercellular Conjugates by Flow Cytometry Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661C6BB1DCEB151C1340A080226633&objectid=6674F2EBA1D257D06F0E2E6B88848643

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

The more commonly available single-laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single-laser machines are used. This protocol describes the labeling of cells and analysis of conjugates with either dual-laser or single laser flow cytometers. - [Read Measurement of Intercellular Conjugates by Flow Cytometry Protocol]

Measurement of Intracellular Ions by Flow Cytometry Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661C4C9AC9935B31A1C2087E2276A3&objectid=6674EF32EE59A37C48B4A267D733CAE2

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

Describes flow cytometric protocols using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer. - [Read Measurement of Intracellular Ions by Flow Cytometry Protocol]

Measuring Apoptosis and Necrosis by Dual Laser Flow Cytometry Protocol - http://cyto.mednet.ucla.edu/Protocols/simultta.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Protocol for measuring apoptosis and necrosis by dual laser flow cytometry. Includes: Preparation of stock solutions of HO342, PI, and 7-AAD; Staining for detection of apoptosis; - [Read Measuring Apoptosis and Necrosis by Dual Laser Flow Cytometry Protocol]

Measuring Apoptosis and Necrosis by Dual Layer Flow Cytometry - http://cyto.mednet.ucla.edu/Protocols/simultta.htm

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Measuring Apoptosis and Necrosis (Oncosis) by Dual Layer Flow Cytometry - [Read Measuring Apoptosis and Necrosis by Dual Layer Flow Cytometry]

Membrane Potential Analysis by Flow Cytometry Protoocol - http://www.niehs.nih.gov/research/atniehs/core/fc/protocols/membrane.cfm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol for membrane potential analysis by flow cytometry. Includes: Preparation of the dyes; Loading of the dyes; Flow Cytometric Analysis. - [Read Membrane Potential Analysis by Flow Cytometry Protoocol]

Minimizing Background in Immunofluorescence Procedures - http://cyto.mednet.ucla.edu/Protocols/minimizi.htm

Category: Molecular Imaging: Immunofluorescence Microscopy

Minimizing Background in Immunofluorescence Procedures. Includes tips and methods on reducing background staining. Janis V. Giorgi Flow Cytometry Laboratory, UCLA. - [Read Minimizing Background in Immunofluorescence Procedures]

Overview of Flow Cytometry - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661BFDC3F5C6C63380B3829E2D6599&objectid=6674E52FF6A6D9943372F453033D787C

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Provides an overview of terminology and echniques unique to flow ytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation-oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry. - [Read Overview of Flow Cytometry]

Packing Capillary Columns for RP-HPLC Protocol - http://cshprotocols.cshlp.org/cgi/content/abstract/2006/28/pdb.prot4546

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. - [Read Packing Capillary Columns for RP-HPLC Protocol]

Pediatric Flow Cytometric Enumeration of Apoptotic Cells PDF. - http://pactg.s-3.com/pub/download/imm/PedsApo.pdf

Category: Cell Biology and Cell Culture: Apoptosis Protocols: TUNEL assay apoptosis

Pediatric Flow Cytometric Enumeration of Apoptotic Cells Using the Tunel Assay. PDF. Nichols et al. PedsAPO - [Read Pediatric Flow Cytometric Enumeration of Apoptotic Cells PDF.]

Phenotype-Specific Immunodetection of Cyclins using 488/630 nm Dual Laser Flow Cytometry Protocol - http://www.cyto.purdue.edu/flowcyt/research/cytotech/telford/cyclind.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Protocol for phenotype-specific immunodetection of cyclins using 488/630 nm dual laser flow cytometry. 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. - [Read Phenotype-Specific Immunodetection of Cyclins using 488/630 nm Dual Laser Flow Cytometry Protocol]

Phycoerythrin Conjugation Protocol - http://pingu.salk.edu/flow/protocols/pelabel.html

Category: Immunology

Protocol for phycoerythrin conjugation. Includes: Preparation of PE; SPDP modification of PE; SMCC modification of antibody; DTT treatment of SPDP-PE; Purification of reactants; Conjugation; Stop reaction; Concentrate product; Separate conjugate. - [Read Phycoerythrin Conjugation Protocol]

Preparation of Cells and Reagents for Flow Cytometry Protocols - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661C3CC685613447CEC768412FDE0F&objectid=6674EA50F5C3D9A0189B376C7A89BF6B

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Preparation of Yeast Cells for Flow Cytometry Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4178

Category: Yeast Protocols: Yeast Nucleic Acid Protocols

Flow cytometry is used to analyze the quantity of DNA in cells. Since the DNA content of cells varies through the cell cycle, this information can provide an indication of cell cycle progression. This protocol uses SYTOX Green staining. - [Read Preparation of Yeast Cells for Flow Cytometry Protocol]

Production of Mouse T Cell Hybridomas Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E663496BA646F5C4FB54D6064E1A19B&objectid=6674C004CF35A9AC863EAD85BD9F080A

Category: Immunology: T Cell Protocols

T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). Protocol describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3-TCR complex by flow cytometry analysis. - [Read Production of Mouse T Cell Hybridomas Protocol]

Propidium Iodide (PI) or DAPI Staining of Unfixed Solid Tissues for Flow Cytometry Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4438

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA in solid tissue samples using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. - [Read Propidium Iodide (PI) or DAPI Staining of Unfixed Solid Tissues for Flow Cytometry Protocol]

Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4437

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA in tissue culture cells using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. - [Read Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry Protocol]

Propidium Iodide Staining of Dead Cells for Flow Cytometry Protocol - http://cyto.mednet.ucla.edu/Protocols/pi.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Propidium iodide (PI) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. - [Read Propidium Iodide Staining of Dead Cells for Flow Cytometry Protocol]

Protocol for Isolation, Cryopreservation, and Thawing of PBMCs - http://www.biomedcentral.com/content/supplementary/1471-2172-6-17-S1.pdf

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cryopreserved PBMCs are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. This protocol standardizes PBMC isolation and cryopreservation techniques, specifically for the assessment of thawed cells by cytokine flow cytometry. - [Read Protocol for Isolation, Cryopreservation, and Thawing of PBMCs]

Protocol for L-selectin-PNAd Interactions under Flow Conditions. - http://www.cbrinstitute.org/labs/springer/protocols/azucena_flow_chamber.html

Category: Cell Biology and Cell Culture

This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]

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Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

This protocol is used in the production of polyclonal antibodies that recognize a peptide sequence of interest.

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.

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