References Library

References Library

In this section will you find tools to learn about the fundamentals of the Technologies available at SCAC - PFCC. We also have available links to Manuals and Standard Operative Protocols that will be valuable for data reproducibility. 

  1. Training Tutorials
  2. Reference Tools
  3. Reference Reading - Publishing Cytometry data & more about the technologies
  4. Manuals and User Guides
  5. Community

Training Tutorials:

These items below provide a basic understanding of flow cytometry.  These highly recommended tutorials are accompanied with pictures and movies, and are excellent for beginners and even advanced users might learn a few things.  

Flow Cytometry basics:  
  1. BD Online Tutorials - Recommended for new users of any flow cytometer: “Introduction to Flow Cytometry”  AND  "A Solid Foundation for Simpler & Smarter Flow Cytometry Panel Design"
  2. Beckman Coulter Online Tutorials - Recommended for new users of any flow cytometer: "Flow Applications" (3) Recommended for new users of any sorter: "The Art of Sorting." 
  3. ISAC CYTO University (FREE): http://cytou.peachnewmedia.com/store/provider/provider09.php
  4. OpenFlow Cytometry initiative (FREE): check YouTube Channel regularly for updated content
  5. ExCyte’s Online Training Webinars: Schedule and access the Core's licenses through PFCC's Core iLab under the training Category. Click HERE to see the available webinars .
SH800 Cell Sorter:

Educational webinar to learn about key aspects of sorting on SH800:

  1. Key Considerations for Everyday Sorting Applications .
Full Spectrum Flow Cytometry:

Learn the basics about the field and before using any of our Full Spectrum instrument by watching these webinars:

cytek aurora

-- MAIN: Cytek's UCSF Full Spectrum Training --

(3 Days session, September 2021 )

FYI Proof of Completion required to register for PFCC's Class 2b. For the specific application of Small Particle/EVs detection see additional required >Webinar below<.

 

Some additional good Full Spectrum references:
  1. "SpectroLearn - Educational Portal" by Cytek
  2. "SpectroFlo Software Tutorials" by Cytek
  3. "UChicago Flow - Cytek Aurora training" by UChicago
  4. "Introduction to Full Spectrum Flow Cytometry" by FlowTex - MSKCC (4/06/2020)
  5. "Foundations for Development of a High Quality Multicolor Full Spectrum Panel" by Cytek (9/28/2020)
  6. "Quality Panel Design to Generate Quality Data with Case Studies" by Cytek (10/12/2020)
  7. "Introduction to Spectral Unmixing" by ChUG Cytometry (5/8/2020)
  8. "Get the FACS—High Order, Hands-On Panel Design" by Stanford FACS Facility (5/21/2020)
Analysis Software
FCS Express

fcs express- UCSF - FCS Express training - (April 2021)

 
 
 
FlowJo

flowjo- UCSF- FlowJo training - (May 2021)

 
 
 
Cytobank: https://www.youtube.com/user/cytobank

 

Reference Tools

Spectral Viewers:

Useful tools for designing multicolor panels and seeing what colors might overlap into each other.  Note, that these spectral viewers are just a guide, and that you should titrate and validate your antibodies every time, to make sure you get optimal staining and are able to identify your population of interest. 

(1) FluoroFinder [NEW]: has available conventional and spectral display

(2) BD   (3) Biolegend   (4) ThermoFisher 

(5) Spectrum by Cytek for spectral analyzers

(6) FPbase for Fluorescent Proteins

For Panel Design:

(7) Fluorofinder with PFCC's instrument configurations

for Antibody Search: (8) BenchSci  and (9) CiteAb

(10) Astrolabe Staining Data Set

OMIPs: find information about published Optimized Multicolor Immunofluorescence Panels for flow cytometry. These can provide you a starting point and alleviate the development time in need of the same or highly similar panels. Use this < ISAC's Public Tableau > for searching purposes.

CellMarker: find information about cell markers of particular cell types in any tissues of interest in human and mouse.

Gaining Insight into Antigen Density:

 

Reference Reading

Publishing Cytometry data

MIFlowCyt Standard by ISAC for Publishing Flow Cytometry data
Data Repositories:
  • FLOW Repository is a database of flow cytometry experiments. It is primarily used as a data deposition place for experimental findings published in peer-reviewed journals in the flow cytometry field
  • ImmPort is funded by the NIH, NIAID and DAIT in support of the NIH mission to share data with the public. 

Flow Cytometry

Practical Flow Cytometry by Howard Shapiro

Great book written about everything you ever wanted to know about flow cytometry.  Free Online PDF version here:  https://www.beckman.com/resources/reading-material/ebooks/practical-flow-cytometry

About Compensation

One of the best pieces written on the subject by Mario Roederer.  View the Informal Perspective, and if you really want to get deep, go through the Detailed Discussion: http://www.drmr.com/compensation/

Flow Cytometry Compensation Tips and Tricks (under 6min)

 
Extracellular Vesicle / Exosomes Detection
  • read published Guidelines: MIFlowCyt-EV framework & MISEV recommendations
  • Publication from Coughlan et al. (2020) Exosome Isolation by Ultracentrifugation
    and Precipitation and Techniques for Downstream Analyses
  • ExoCarta, a curated database of exosome proteins, RNAs, and lipids. 
  • FunRich, a non-commercial software tool, to identify biomolecules that are over-represented in exosomes compared with their levels within originating cells.
  • Additional Resources: http://www.evflowcytometry.org/  and NIH NanoBiology section
  • PFCC recommended webinars on this topic:
1- Entering the submicron world through the gates of best practices by Joanne Lannigan (April 2021) --- required after Class 2b
2- Measuring Single EVs and their cargo
3- Developing Tools for Multiplex to Single EV Analysis Pipelines by Joshua Welsh

Mass Cytometry

CyTOF

(more coming soon)

MIBI

Genomics

Fluidigm

C1 Open App program: customers can create and share single-cell applications with each other. Click HERE for more information

10x Genomics

Getting started with 10x: https://www.10xgenomics.com/solutions/single-cell/#getting-started

Manuals and User Guides

PFCC Manual Standardization SOP

A manual version of standardizing any cytometer for a long term study.  Useful for setting up the instrument in a reproducible manner once a panel has been established.  This would be the manual version of BD's Application Settings.  Please review this SOP with Core staff before utilizing.   

BD Application Settings Standardization Method

BD's automated way of standardizing the instrument.  See LSR/Fortessa or Aria Guides further down this list for the Quick Reference guides regarding Application Settings. Please contact Core staff before using Application Settings as the instrument's baseline cannot be reset for Application Settings to work properly.  The following two items discuss an overall quick summary as well as detailed information regarding BD's Application Settings:  1) Quick Application Settings Guide  2) Detailed Application Settings Bulletin

FACSCalibur Guides

These items below are useful reference guides after receiving FACSCalibur hands-on training.

Official manuals from BD for the FACSCalibur as well as focus areas on the following: 1) CellQuest Pro Software for Analysis and Acquistion for users 2) FACSComp for service  

LSR/Fortessa/X-20/HTS Guides

These items below are useful reference guides after receiving LSR/Fortessa/X-20/HTS hands-on training.

About Application Settings:

FACSDiva Software Quick Reference Guide for setting up & utilizing Application Settings.  It is an automated method of machine standardization for long term studies for controlling the instrument's day to day changes.  Please review with Core staff before utilizing Application Settings or consider using the PFCC manual method listed under Instrument Standardization Guides above.   

Calcium Flux: Guide for optimizing Calcium Flux assays

Official manuals from BD for the following:

1) LSRII 2) Fortessa/X-20 3) HTS for users 4) HTS for service

FACSAria Guide - Application Settings:

FACSDiva Software Quick Reference Guide for setting up & utilizing Application Settings.  It is an automated method of machine standardization for long term studies for controlling the instrument's day to day changes.  Please review with Core staff before utilizing Application Settings or consider using the PFCC manual method listed under Instrument Standardization Guides above.    

FACSAria Guide - Plate and Slide Sorting:

Guide for using the ACDU to sort onto a plate or slide.

Manuals:

Official manuals from BD for the following 1)  AriaII 2)  AriaIII 3)  AriaFusion

Community

Best Listserv to join

The Purdue PUCL Cytometry discussion. Established in 1989, the list has been continuously available for free access to all scientists:  http://www.cyto.purdue.edu/hmarchiv/index.htm

Northern CA Flow User Group Meeting: norcalcyto.org

International Society for Advancement of Cytometry: https://isac-net.org/