Resources

General

How to use FlowJo off-campus using an Emory VPN connection

Follow these instructions to create and open a VPN (Virtual Private Network) connection to the Emory system.

Then fire up FlowJo (make sure you've registered FlowJo!) Let us know if you have any questions.

How to connect to the Flow Core server for data transfer

• Instructions for Macintosh (AppleShare IP)
• Instructions for Windows (SAMBA)
• Instructions for OS X

Protocols and Procedures

• Antibody Titering(Carleton Stewart, et al)
• Immunophenotyping (Carleton Stewart, et al)
• Cell Cycle Staining Protocol (special Thanks to Dr. Ritu Aneja and the Harish Joshi Lab)
• A WinMDI Tutorial
• A Setup System for Compensation: BD CompBeads plus BD FACSDiva Software (pdf)
• BD FACSAria User's Guide
• Determining Antibody Titers (special Thanks to Dr. Carleton Stewart and RPCI)
• Titering Directly Conjugated Antibodies to Extracellular Antigens (special Thanks to Dr. Carleton Stewart and RPCI)
• Apoptosis Prep for Training
• Extracellular Immunophenotyping prep (special Thanks to Hilary Rosenthal, MT (ASCP) and the Waller Lab)
• Intracellular Staining prep (special Thanks to Hilary Rosenthal, MT (ASCP) and the Waller Lab)
• Cell-cycle protocol for yeast (special Thanks to Missy Brykailo and the Fridovich-Keil Lab)

Training Materials

• Post-Training Tutorial - Digital Analyzers
• Post-Training Tutorial - Analog Analyzers
• DSB LSRII Reagent/Filter/Laser Chart
• DSB LSRII Octagon Optic Template
• DSB LSRII Trigon Optic Template
• DSB LSRII panel worksheet (special thanks to Suzanne Mertens and the EVC  Flow Cytometry Core Facility)

Sorting

In order to avoid the common pitfalls inevitable in preparing cells for sorting, we recommend reading the following links:

• Guide to Getting a Great Sort
• Sort Buffer Selection
• BD FACService Technotes, Vol 9 No. 4, Oct 2004

Sorting Presentations

• Source Lab PI: Dr. Rafi Ahmed, PhD
• Client: Dr. Kim Kawamura, PhD
• Source material: P14 chimeras (B6 mice initially transferred with 106 P14 splenocytes) and then immunized with DNA
• Sort criteria: separate out CD62L hi and lo populations from DNA- immunized mice 62 days post-vaccination
• Sort goal: to use the sorted cells to make RNA for gene chip analysis
• Sort protocol: high-speed two-step sort

References

1. Flow Cytometry: A Practical Approach. Edited by MG Ormerod. ( IRL Press, Oxford. 1994. ISBN 0-19 963461-0)
2. Practical Flow Cytometry. 3rd Edition. Howard M Shapiro. ( Alan R Liss, Inc. ISBN 0-471-30376-3).
3. Flow Cytometry. First Principles. Alice Longobardi Givan. ( Wiley-Liss, New York, 1992. ISBN 0-471-56095-2.)
4. Handbook of Flow Cytometry Methods. Edited by J Paul Robinson. ( Wiley-Liss, New York, 1993. ISBN 0-471-59634-5. )

Detailed facility information is given below. If you need additional information please contact the Core Director.

Instrumentation

The Emory University School of Medicine Core Facility for Flow Cytometry is located in the Dental School Building. Support in the form of training, technical assistance, troubleshooting and consultation are given to various laboratories that independently own cytometers on campus. Facility hardware:

All A3 analyzers have been configured with 5 lasers and 25 detectors (23 colors + 2 scatter) ; sorters have been configured with 5 lasers and 19 detectors (17 colors + 2 scatter). The laser wavelengths are 355nm (UV), 407nm (violet), 488nm (blue), 561nm (yellow-green), 633nm (red).

Analyzers:

• Symphony A3 (Location: Dental School, Room 426)
• Symphony A3 (Location: RRC 3185)

Sorters:

• FACSAria IIA (Location: Dental School, room 441)
• FACSAria IIB (Location:  Dental School, room 441)