Flow Cytometry (FCRG)

Mission

The Flow Cytometry Research Group was formed in 2012 by Peter Lopez (NYU) and Scott Tighe (UVM) with the goal of providing information related to the art of flow cytometry, including its cross-technology applications to genomics, proteomics, and other core-related areas.

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Current Membership 

Mehrnoosh Abshari, NIH/NIDCR
Roxann Ashworth, Johns Hopkins University, School of Medicine, EB Liaison
Claudia Bispo, University of California San Francisco
Sara Bowen, Dignity Health St. Joseph's Hospital and Medical Center
Ching-Yuan (Steve) Chen, Columbia University Irving Medical Center
Kevin Ferro, Stowers Institute for Medical Research
Christiane Hassel, Indiana University
Pam Moody, Cold Spring Harbor Laboratory
Kathy Schaefer, HHMI Janelia Research Campus, Co-Chair
Rachael Sheridan, Van Andel Research Institute Flow Cytometry Core
Celine Silva-Lages, Cincinnati Children’s Medical Center
Jane Srivastava, Gladstone Institutes, Co-Chair
John Tigges, Beth Israel Deaconess Medical Center
Eric Wieder, University of Miami Miller School of Medicine

Summary

The Flow Cytometry Research Group (FCRG) focuses on flow cytometry-related projects. In many experimental workflows, cells sorted in a flow cytometry core are further analyzed in other cores. Currently, the RG emphasizes exploration of the various effects that cell sorting may or may not have on sorted material and thus the effects on downstream analysis by other shared facilities.

CURRENT STUDY

DROP DELAY

It is known that correct drop delay calibration and accurate sort recovery is always critical when sorting. The FCRG's current study will test drop delay settings in sorters with options to set drop delay manually and those with fully automated, non-adjustable drop delay set-up. Data collected will help to determine the accuracy of drop delay and sort recovery and purity for both types of sorters, and help serve as a guide for sort operators to assist in determining drop delay accuracy.


PAST STUDIES/PROJECTS

GENE EXPRESSION AFTER SORTING

The first study proposed by the group investigated the impact of cell sorting on gene expression. Variables such as sorting versus no sorting, high versus low pressure, and the presence versus absence of UV light were analyzed with RNA-seq and microarray in Jurkat cells, primary B cells, and mouse ES cells. A manuscript with this study’s results is being prepared.

SORTER CLEANLINESS

Cytometry facilities around the world were surveyed regarding their sorter cleaning practices. A subset of facilities submitted samples for testing. Most sorters had significant endotoxin contamination, but little to no RNase. Bacterial concentrations ranged from none to substantial. There was no correlation found between sorter cleanliness and any surveyed facility variables, including sorter age, cleaning practices, date of last PM, sheath source, or known recent contamination. The results will be published in combination with the endotoxin removal results.

ENDOTOXIN REMOVAL

Since a number of sorters assayed in the sorter cleanliness study were contaminated with endotoxin, effectiveness of an H2O2 cleaning procedure was tested to assess removal of the endotoxin from the sorters. Sheath samples collected before and after cleaning were tested with a LAL quantitation kit and it was determined that the contamination was only partially mitigated. Also, the endotoxin levels reached pre-cleaning levels within a few weeks of the sterilization. The results will be published alongside the sorter cleanliness survey results.

Presentations and Posters

  1. ABRF 2019 poster - A Multi-Core Study on How Different Fixation Methods Prior to Sorting Impact the Purity, Quality, and Yield of RNA From Sorted Cells
  2. ABRF 2018 Scientific Session - Single Cell Sorting and the Bioinformatics Pathway
    1. The Flow Cytometry Research Group: A (Roughly Half) Decade in Review by Dave Adams
    2. Defining Novel Multilineage Progenitor Populations Using Single-Cell RNA-Seq by Nathan Salomonis
  3. ABRF 2018 Satellite Workshop - Bridging the Gap: Isolation to Translation (Single-Cell RNA-Seq)
    1. Basics of RNA-Seq by Michael Kelly
    2. Methods, Applications & Analysis of scRNA-Seq: How an Integrated Understanding of Every Step Makes for a Better Single Cell Experiment by Michael Kelly
    3. Open Workflows to Enable and Understand Single-Cell RNA-Seq Data by Nathan Salomonis
  4. ABRF 2017 poster - Endotoxin Contamination of Cell Sorters: Evaluating Cleaning and Testing Procedures
  5. ABRF 2017 - Sheath Contamination Survey: An Examination of Common Laboratory Practices
    Presentation by Dave Adams
    Poster
  6. ABRF 2016 - Evaluating the Effects of Cell Sorting on Gene Expression
    Presentation by Andrew Box
    Poster
  7. ABRF 2016 poster - Evaluating Cell Sorter Cleaning Procedures Across ABRF-FCRG Institutions by Testing for Common Contaminants 
  8. ABRF 2015 - Evaluating the Effects of Cell Sorting on Gene Expression
    Presentation
    Poster
  9. ABRF 2014 poster - Evaluating Effects of Cell Sorting on Cellular Integrity and Gene Expression
  10. ABRF 2013 presentation by Scott Tighe - Overview of the New FCRG and Proposed Cell Sorting (FACS) Microarray Study
  11. ABRF 2013 poster - The New ABRF Flow Cytometry Research Group

Protocols

1) This paper has a detailed protocol for how to properly prepare a sorter for RNA isolation and sort cells with Trizol, RLT, and RNase inhibitor. It also includes an estimated yield of RNA from certain cell types.
    - View Document (802K)

Membership History

Member NameOrganizationDetails
Andrew C. Box Stowers Institute for Medical Research  Member: 11/12 - 03/16
Co-chair: 05/15 - 03/16
Ad hoc: 04/16 - 04/21
Dr Sridar V Chittur SUNY Albany  Member: 11/12 - 05/15
Monica DeLay CCHMC  Member: 09/12 - 03/17
Co-chair: 03/13 - 03/16
Ad-hoc: 04/17 - 04/21
Maris Handley Massachusetts General Hospital Member: 04/15 - 03/17
Peter Lopez NYU Office Collaborative Science  Member: 09/12 - 03/15
Ad hoc: 04/15 - 04/21
Dr Thomas Neubert New York Univ Sch of Med  EB Liaison: 09/12 - 03/13
Mr. Hank Pletcher University of Pennsylvania  Ad hoc: 11/12 - 03/13
Scott Tighe Vermont Cancer Center  Chair- RG Co-Founder: 02/12 - 03/13
Member, ad hoc-co-chair: 04/13 - 05/15
Paula B Turpen University of Nebraska Medical Center  EB Liaison: 04/13 - 04/14
Frances Weis-Garcia    Memorial Sloan Kettering Cancer Center    EB Liaison: 05/14 - 03/17

 

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