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Human Immune Monitoring Shared Resource Equipment and Services


EXPERIMENTAL DESIGN SUPPORT

The HIMSR intends to fill in gaps between clinical and basic science research, providing sample preparation and immune-based assays that fit the needs of our campus and scientific community and integrate with existing core services. Contact us to tell us what you might need from us in the future to help inform our ongoing assay development and validation process!

To better serve our campus, we offer free initial experimental design support regarding research endpoints, the selection of appropriate assays, the coordination of investigators with available statisticians and bioinformaticians to assist in study design and data analysis, and to test and develop new technologies for immune monitoring.
 
Standard services: Available on a fee-for-service basis (see iLabs request form or contact Kim Jordan)
Customized projects: Quotes are available.  For projects involving ongoing experimental design support, assay development, or extensive analysis, we can also discuss percent effort contributions. 
 
A goal of the HI3 is to foster relationships across disciplines to better leverage our human sample resources.  Please contact Aimee Bernard (Aimee.Bernard@ucdenver.edu) to learn how you can contribute to this effort and to add to our growing list of research needs.

SAMPLE PROCESSING

Blood cell isolation

We offer isolation of leukocytes from whole blood using ficoll-paque gradient separation, CPT tubes, Accuspin tubes, red blood cell lysis, or other protocol-specific isolation methods.  Isolated cells can then be used immediately in downstream assays, or frozen and stored for future analysis.  These samples can be temporarily stored in our facility until co​​​mpletion of the study and/or experimental procedures.


Smart Tubes and the Smart Tube Base Station
Located in the Hsieh Lab as an extension of the HI3 HIMSR
The Smart Tube system is a compact, fully automated system that simplifies whole blood processing and subsequent analysis while ensuring an unparalleled level of consistency and reproducibility. Simply add freshly collected whole blood to the Smart Tube, insert them into the Smart Tube Base Station instrument and, at the end of the automated prep, the samples can be directly placed in the freezer for future analysis. The Smart Tube system reduces variability in sample processing and our programmable station can be used to program other Smart Tube base stations at other locations within a multi-center study http://www.smarttubeinc.com/


Tissue cell isolation

 

Miltenyi gentleMACS - COMING SOON!
We will offer the isolation of tissue infiltrating leukocytes using Miltenyi’s gentleMACS dissociation system.  https://www.miltenyibiotec.com/en/products-and-services/macs-sample-preparation/sample-dissociation.aspx
 
Plasma/Serum Preparation
We offer plasma or serum collection from blood tubes, division into appropriately sized aliquots for downstream assays, and temporary storage until completion of the study and/or experimental procedures.
 
RNA/DNA isolation 
We will prepare RNA or DNA after any isolation procedure (e.g. PBMC isolation, cell sorting, etc.) and help coordinate downstream analysis.

CELL SORTING


BD FACSAria Fusion
Located in the CU Department of Immunology HI3 HIMSR Flow Facility
Depending on your needs, we can provide full service antibody panel design, cell staining, and cell sorting, or sort cells stained in your own laboratory by Fluorescence-Activated Cell Sorting on the BD FACSAria Fusion, https://www.bdbiosciences.com/us/instruments/research/cell-sorters/bd-facsaria-fusion/m/1526412/overview.


 
Miltenyi AutoMACS- COMING SOON!
We will offer full service labeling and separation of leukocyte populations using Miltenyi’s magnetic bead-based autoMACS separator.  http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation.aspx

CYTOMETRY
Mass Cytometry
 
Fluidigm Helios CyTOF
Located in the CU Cancer Center Flow Cytometry Core
Mass cytometry, or cytometry by time-of-flight (CyTOF®), enables enhanced comprehension by simultaneously measuring more than 40 parameters for millions of cells using high-purity heavy metal isotope tags to ensure minimal background from signal overlap or endogenous cellular components. Interested? See the different levels of services offered to perform these experiments under ‘Rates and Fee Structure’.
 
Standardized CyTOF staining panels and analysis - COMING SOON!
We are developing standardized immune cell marker panels and will offer sample preparation, stimulation, barcoding, and staining as a fee-for-service option.  These samples will be handed off to the CyTOF experts in the CU Cancer Center Flow Cytometry Core for acquisition.  Depending on the investigator’s needs, the HIMSR will also offer analysis services using Cytobank (described below) and provide conjugation and verification services for antibodies labeled with heavy metals

Cytobank
Located in the HI3 HIMSR
The secure cloud-based Cytobank analysis platform provides an array of analysis tools, including dot plots, clustering and dimensionality reduction algorithms (SPADE,viSNE, and Citrus), and summary statistics tools like heat maps and dose-response curves that transform data into high-impact knowledge. Please contact elena.hsieh@ucdenver.edu for information about the HI3-sponsored enterprise account, a full-service analysis and secure storage for users on our campus.
 
 
Flow Cytometry
 
BD FACSCalibur
Located in the CU Department of Immunology & Microbiology HI3 HIMSR Flow Facility
The BD FACSCalibur is a multicolor benchtop flow cytometry system. It is equipped with 2 lasers and capable of running samples labeled with up to 4 colors (FITC, PE, PerCP-Cy5.5, APC). Different levels of service are offered under ‘Rates and Fee Structure’.

 
BS LSR Fortessa X-20
Located in the CU Department of Immunology HI3 HIMSR Flow Facility
The BD LSR Fortessa X-20 instrument delivers the optimal sensitivity and resolution required for multicolor applications and has 5 lasers that can be used to detect up to 18 colors simultaneously. Different levels of service are offered under ‘Rates and Fee Structure’. (https://www.bdbiosciences.com/us/instruments/research/cell-analyzers/bd-lsrfortessa-x-20/m/1519232/overview)
 
Standardized flow cytometry staining panels - COMING SOON!
We are currently developing standardized antibody panels for the analysis of human immune cells using the Fortessa instrument and will soon offer them as a fee-for-service option.  These panels will include immune cell phenotyping markers, functional markers, intracellular cytokines, phospho-proteins, and antibodies/Ig-fusion proteins for CAR-T cell detection.  We will also provide these antibody clone names and staining protocols for investigators wishing to perform the staining in their own laboratories. 
 
Contact us now to get your favorite markers included in this development phase!

IMAGING
 
Vectra 3
Located in the HI3 HIMSR
The Vectra 3 is an automated quantitative pathology imaging system that detects and distinguishes up to seven stains (6 markers plus DAPI or counterstain) on slides imaged in brightfield (H&E, IHC) or immunofluorescence (IF) using intact tissue microarray (TMA) or formalin-fixed, paraffin-embedded (FFPE) tissue sections.  We are currently offering slide scanning, please contact Kimberly.jordan@ucdenver.edu for more information. http://www.perkinelmer.com/product/vectra-3-0-200-slide-cls142338

 
inForm
Located in the HI3 HIMSR
inForm is a patented automated image analysis software package that uses trainable algorithms in a user-friendly interface to accurately visualize and quantify biomarkers in tissue sections.  Capability includes phenotyping, co-localization, co-expression, tissue region detection (e.g. tumor versus stroma), and traditional scoring functions.  This software is available for use on a dedicated analysis station within the HI3 HIMSR.
 
Standardized IF slide staining - Leica Bond RX Autostainer
We have developed standardized antibody panels for the analysis of immune cell infiltrate in human FFPE tissue and offer them as a fee-for-service option.  These panels include basic immune cell phenotyping markers (CD4, CD8, FOXP3, CD3, CD68, CD56, CD19, CD20, CD11b, CD11c, CD33, CD14, CD15, CD163, CSFR1, CD66b, and elastase), inhibitory markers (PD-1, PDL1, LAG-3, TIM-3, IL-10, CTLA4, IDO), activation/effector molecules (CD45RO, HLADR, Ki67, Granzyme B, IL-1β, and tissue markers (pan-cytokeratin, SOX10, alpha-actinin, smooth muscle actin, and EPCAM).  These panels utilize secondary polymer and TSA reagents produced by Perkin Elmer that are optimized for detection by the Vectra instrument.  We will also provide the antibody clone names and staining protocols for investigators wishing to perform the staining in their own laboratories.  
 
Custom IF antibody panel development
If a standard panel is not available for your markers of choice, we would like to work with you to develop a custom antibody panel either in your lab or the HIMSR.  In the spirit of fostering collaborative research at CU AMC, antibody panels developed in the HIMSR will be shared with the campus as future standard panels.

IMMUNOASSAYS
 
Meso Scale Discovery (MSD) Instrument – COMING SOON!
Located in the HI3 HIMSR
An automated MSD instrument that uses electrochemiluminescence technology to provide unparalleled sensitivity and dynamic range for the detection of up to 10 analytes in each immunoassay well. This automated instrument requires low sample volume, eliminates human error, and greatly reduces variability.  HIMSR will provide full-service options from collection and storage of biologic fluids, to running, reading, and analysis of immunoassays.  All V-Plex assays will be available immediately after instrument installation as standard fee-for-service assays (https://www.mesoscale.com/en/products_and_services/assay_kits/v-plex/v-plex_assay_menu), with other capability to follow soon after (U-Plex, PK Assays, and Immunogenicity Assays). 

 
Luminex
Located in the CU Cancer Center Flow Cytometry Core
We will perform full-service immunoassays using Luminex multiplex magnetic bead array platform.  Assays performed in the HIMSR will be read and analyzed in the CU Cancer Center Flow Cytometry Core.
 
ELISA – COMING SOON!
Single analyte ELISAs, purchased and performed as needed.

IMMUNE FUNCTION
Ex vivo stimulation - COMING SOON!
Isolated leukocytes can be stimulated ex vivo with specific peptide or antigen mixtures, non-specific stimuli (PHA, PMA/ionomycin, antibodies, bead-bound antibodies, cytokines, LPS, etc), or allogenic cells (mixed lymphocyte reactions).  Cellular function can then be assessed +/- stimulation in proliferation assays, flow or mass cytometry for intracellular cytokines or phosphorylated signaling molecules, ELISA of assay supernatants, or ELISPOT.  
 
ELISPOT - COMING SOON!
We will offer this method for determining cellular function after stimulation in specialized plates coated with antibodies specific for the secreted factor of interest (e.g. IFNγ, TNFα, etc).

 
Smart Tubes and the Smart Tube Base Station
Located in the Hsieh Lab as an extension of the HI3 HIMSR
The Smart Tube system is a compact, fully automated system that simplifies whole blood processing and subsequent analysis while ensuring an unparalleled level of consistency and reproducibility. Simply add freshly collected whole blood to the Smart Tube, insert them into the Smart Tube Base Station instrument and, at the end of the automated prep, the samples can be directly placed in the freezer for future analysis. http://www.smarttubeinc.com/

DATA ANALYSIS
 
inForm
Located in the HI3 HIMSR
inForm is a patented automated image analysis software package that uses trainable algorithms in a user-friendly interface to accurately visualize and quantify biomarkers in tissue sections from the Vectra 3.  Capability includes phenotyping, co-localization, co-expression, tissue region detection (e.g. tumor versus stroma), and traditional scoring functions.  This software is available for use on a dedicated analysis station within the HI3 HIMSR. http://www.perkinelmer.com/product/inform-cell-analysis-one-seat-cls135781
 
Spotfire
Located in the HI3 HIMSR
Spotfire (TIBCO) platform assimilates data from multiple sources, enables data visualization, and allows complex analyses in an easy-to-use visual tool.  inForm data integrates well with this platform for higher-level analyses.  This software is available for use on a dedicated analysis station within the HI3 HIMSR. http://spotfire.tibco.com/
 
Cytobank
Located in the CU Department of Immunology HI3 HIMSR Flow Facility
The secure cloud-based Cytobank analysis platform provides an array of analysis tools, including dot plots, clustering and dimensionality reduction algorithms (SPADE,viSNE, and Citrus), and summary statistics tools like heat maps and dose-response curves that transform your mass cytometry data into high-impact knowledge. Please contact Elena Hsieh (elena.hseih@ucdenver.edu ) for information about the HI3-sponsored enterprise account, a full-service analysis and secure storage for users on our campus.  This software is available for use on a dedicated analysis station in the CU Department of Immunology HI3 HIMSR Flow Facility. https://www.cytobank.org/
 
FlowJo
Located in the CU Department of Immunology HI3 HIMSR Flow Facility
The FlowJo™ workspace is a powerful statistical environment that is used for a variety of research needs, including immunophenotyping, cell cycle, proliferation, kinetics studies, quantitative population comparison, and plate screening assays.  This software is available for use on a dedicated analysis station in the CU Department of Immunology HI3 HIMSR Flow Facility. https://www.flowjo.com/

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