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.
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.
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.
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.
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
- ABRF 2018 Scientific Session - Single Cell Sorting and the Bioinformatics Pathway
- Defining Novel Multilineage Progenitor Populations Using Single-Cell RNA-Seq by Nathan Salomonis
- ABRF 2018 Satellite Workshop - Bridging the Gap: Isolation to Translation (Single-Cell RNA-Seq)
- Open Workflows to Enable and Understand Single-Cell RNA-Seq Data by Nathan Salomonis
- ABRF 2017 poster - Endotoxin Contamination of Cell Sorters: Evaluating Cleaning and Testing Procedures
- ABRF 2017 - Sheath Contamination Survey: An Examination of Common Laboratory Practices
Presentation by Dave Adams
- ABRF 2016 - Evaluating the Effects of Cell Sorting on Gene Expression
Presentation by Andrew Box
- ABRF 2016 poster - Evaluating Cell Sorter Cleaning Procedures Across ABRF-FCRG Institutions by Testing for Common Contaminants
- ABRF 2015 - Evaluating the Effects of Cell Sorting on Gene Expression
- ABRF 2014 poster - Evaluating Effects of Cell Sorting on Cellular Integrity and Gene Expression
- ABRF 2013 presentation by Scott Tighe - Overview of the New FCRG and Proposed Cell Sorting (FACS) Microarray Study
- ABRF 2013 poster - The New ABRF Flow Cytometry Research Group
|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)
|Andrew C. Box||Stowers Institute for Medical Research||Member: 11/12 - 03/16|
Co-chair: 05/15 - 03/16
Ad hoc: 04/16 - present
|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 - present
|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 - present
|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 CoFounder: 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|