The CSF provides Fox Chase investigators with access to state-of-the-art flow cytometry equipment and expertise. Major applications of flow cytometry include discrimination of biomarkers on cells and isolation of subpopulations of single cell suspensions by sorting at up to 30,000 cells/second. Examples of cells that can be purified by flow cytometry-based sorting include leukocytes, fluorescent protein-expressing cells, and rare variant cell subpopulations. Flow cytometry is useful for single cell cloning of cell lines by direct deposition into 96-well plates. The CSF has two primary components: Research Flow Cytometry and Human Immune Monitoring.
Research Flow Cytometry Services
Research Flow Cytometry provides support for basic research laboratories that primarily analyze cell lines and cells from mouse models. Analysis of biomarkers can include characterization of surface or intracellular marker expression on tumors or cell lines with fluorophore-conjugated antibodies and measurement of intracellular calcium flux. The facility also aids users with advice on experimental design (including availability of reagents and appropriate fluorochromes), sample preparation, and data interpretation. Investigators are trained to operate the BD FACScan and LSR-II flow cytometers without the need for a dedicated operator, making scheduling on these instruments very flexible. For large-scale cell separations, pre-fractionation of cells prior to electronic cell sorting by use of magnetic bead technology is also available using a Milteny MACS device in the facility. Data from all instruments can be annotated, collected, and managed for long-term storage using a Flow-LIMS, developed in-house together with the Bioinformatics Facility. A networked flow cytometry analysis application (FlowJo program) runs on individual users’ laboratory computers and provides highly interactive and graphical analysis of multiparameter data. The standardization of approaches through the CSF, coupled with the Flow-LIMS data warehouse, facilitates collaboration and the sharing of data, which accelerates discovery.
Flow cytometry analysis
Cell surface staining using fluorescent-conjugated monoclonal antibodies can be analyzed. Internal determinants can be analyzed if cells are first fixed and permeabilized prior to staining.
Flow cytometry sorting
Cells with desired characteristics (including distinctive biomarker expression or GFP+) can be isolated in up to four separate streams simultaneously under sterile conditions.
Intracellular calcium flux measurement
Measurement of Indo-1-AM-loaded cells enables determination of intracellular calcium ion concentration and alteration after cell stimulation.
Assistance with experimental design
The CSF provides information on uses of flow cytometry and cell sorting for the user community. Personnel in the CSF provide expert advice regarding experimental feasibility, such as the number of cells that can reasonably be isolated per day or the assembly of complex staining panels.
Human Immune Monitoring Services
The Human Immune Monitoring component of the CSF processes fresh blood, bone marrow, or lymph node samples from patients or healthy human donors and analyzes these samples for expression of numerous immune biomarkers using 14-color multiparametric flow cytometry. In addition, high-purity flow cytometry-based sorting of immune cell subpopulations or tumor cells is performed for further studies (functional assessment, genetic analysis, etc.). Ongoing correlative studies are determining changes in immune phenotype and function between pre- and post-treatment samples from patients on immunotherapy clinical trials at Fox Chase and collaborating institutions nationwide. The goals are to identify biomarkers that could predict beneficial responses to immunotherapies in these patients and to identify potential targets for additional immune therapy intervention. In addition, new technologies and assays are being adopted and developed to achieve these goals. Any remaining blood, plasma, and tissue samples are cryopreserved for future studies or passed on for additional molecular studies, including RNA-seq, NanoString, and cytokine quantification in other FCCC Facilities. The Human Immune Monitoring services can be used to validate biomarkers, conduct immune assays, support basic science and/or clinical trials (i.e., testing hypotheses or drug efficacies), and/or generate pre-clinical data to justify future investigator-initiated studies.
Providing cancer immunology expertise
The Human Immune Monitoring component provides full-service expertise to design, guide, and perform correlative immune studies for clinical trials at Fox Chase. Clinicians are guided through the entire process of formulating the correlative research components of clinical trial proposals, establishing an assay strategy, designing antibody staining panels, data analysis, and providing final interpretations of results.
Immune phenotyping of human leukocytes
Multiparameter flow cytometry is used to analyze the phenotype of immune cells (T, B, NK, monocytic, and dendritic cells). Specific assays include standard fluorophore-conjugated antibody staining for cell surface or intracellular biomarkers, including markers for activation, proliferation, and exhaustion, as well as for receptors and subsets of leukocytes. Data analysis can gate on individual subsets of cell types and establish the mean level of expression of a particular biomarker on that subset or the percentage of cells expressing that biomarker.
Assays of T and NK cell functions
Human T and NK cell functional responses can be measured by stimulating the cells under various conditions in vitro and staining for intracellular expression of interferon or surface expression of the degranulation marker CD107a.
Sorting of cell subsets for downstream assays
Up to four subsets of human cells can be simultaneously sorted, based upon surface marker expression profiles. Once isolated in this way, the cells can be subjected to various downstream molecular and functional assays.
Release of immune cells from human tissues for flow cytometry analysis
A Miltenyi gentleMACS Tissue Dissociator unit is used to digest and dissociate single cells from human solid tumor or normal tissue samples for downstream flow cytometry-based analysis or sorting.