Flow Cytometry Lecture

New users are required to take the Flow Cytometry Lecture. Experienced users are encouraged to look through for updates. Download so you can hear my voice.

• Flow Cytometry Theory
• Compensation
• Instrumentation
• Stain Index and Spillover Spreading Matrix on LSR II and Aurora

Xiaoxuan Fan, Director
XiaoxuanFan@som.umaryland.edu 

LSR II Cleaning

• SOP and Tube Catalog #

Sample Prep

• PBMC Staining Procedure
• PBMC Freeze/Thaw
• Sorting Sample Requirements

Guidelines for Publication

• Eur. J. Immunol – Guidelines for Flow Cytometry in Immunological Studies
• Standards Required for Reproducibility and Rigor in Research Flow Experiments
• Compliant Manuscript Using ISAC FCS File Repository (FlowRepository.org)
• J. Immunol – Guidelines for Automated Data Analysis

Compensation and Unmixing Theories

• Mario Roederer: Distributions of autofluorescence after compensation: Be panglossian, fret not

YouTube Flow Channels

• OpenFlow: Compensation, BD Cytometer Operation, Spectral Cytometry
• FlowTex: Excellent presentations at annual meetings
• Perspectives on spectral flow cytometry: Babraham Institute

Cytek Aurora

• Cytek Tutorial and Resources
• Theory and Operation (Part I, Part II)
• Aurora Panel Design Template (Please use this table format to communicate with flow core. Dr. Fan's 23, 29 color panels are here)
• Customizable mouse backbone spectral panel
• 4 Laser Aurora Fluorochrome Guide
• BioLegend Multicolor Panel Selector
• LSR II and Aurora stain index and spillover comparison
• Plate Loader SOP
• FlowJo settings

Spectral Cytometry Panel Publications

• Full spectrum flow cytometry and mass cytometry: A 32-marker panel comparison
• Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry
• OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood

Optimized Multicolor Panels

• OMIPs from Cytometry A

Technical Note

• Area Scaling with FACS DIVA

Apoptosis

• Cytometry in Cell Necrobiology Revisited. Cytometry A 2010
• Apoptosis and beyond: cytometry in studies of programmed cell death. Methods Cell Biol. 2011
• Measurement and Characterization of Apoptosis by Flow Cytometry. Curr. Protoc. Cytom. 2016 (full text through library)

DNA Staining

• Analysis of Cellular DNA Content by Flow Cytometry
• YouTube Tutorial: Cell Cycle Analysis using FCSExperss 7 (Important to use Mathematical Models)

Proliferation Assay

• The Use of CFSE to Monitor Lymphocyte Proliferation. J. Vis. Exp. 2010 (video)
• Monitoring Lymphocyte Proliferation with CFSE. Nat. Protoc. 2007
• Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Current Protoc. Cytom. 2013

Can request through the library. This one includes the alternative dye, Cell Trace Violet, less toxic and free up the FITC/GFP detector. 

Extracellular Vesicles

• Reliable measurements of extracellular vesicles by clinical cytometry
• MIFlowCyt-EV Standardized Procedure

Imaging Cytometry

• Overview of Unique Features (YouTube video)
• IDEAS Software