Flow Cytometry (FCRG)

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.

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.

Poster presented at 2023 CYTO and ABRF Annual Meetings:

PAST STUDIES/PROJECTS

FCRG Community Survey (2021)

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.

1. ABRF 2022 workshop - Caveats and Best Practices for Study Design and Data Analysis in High-dimensional Flow
2. 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
3. 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
4. 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
5. ABRF 2017 poster - Endotoxin Contamination of Cell Sorters: Evaluating Cleaning and Testing Procedures
6. ABRF 2017 - Sheath Contamination Survey: An Examination of Common Laboratory Practices
   Presentation by Dave Adams
   Poster
7. ABRF 2016 - Evaluating the Effects of Cell Sorting on Gene Expression
   Presentation by Andrew Box
   Poster
8. ABRF 2016 poster - Evaluating Cell Sorter Cleaning Procedures Across ABRF-FCRG Institutions by Testing for Common Contaminants 
9. ABRF 2015 - Evaluating the Effects of Cell Sorting on Gene Expression
   Presentation
   Poster
10. ABRF 2014 poster - Evaluating Effects of Cell Sorting on Cellular Integrity and Gene Expression
11. ABRF 2013 presentation by Scott Tighe - Overview of the New FCRG and Proposed Cell Sorting (FACS) Microarray Study
12. ABRF 2013 poster - The New ABRF Flow Cytometry Research Group