Center for Vaccine Research

Shared Resources

The Center for Vaccine Research (CVR) Members and Affiliates have access to a variety of cutting edge technologies. For questions regarding CVR resources, please contact the CVR’s Director of Operations, Dr. Jodi Craigo, craigoj@pitt.edu or 412-624-4610.

 

FLOW CYTOMETRY

The CVR has analytical and sorting capabilities in biosafety level 2-3 facilities. We have an analytical machine in our BSL2 facility, two cell sorters, one in BSL3 and the other in BSL2+ as well as an AutoMACS Pro for lower specificity cell isolations or specific cellular population enrichments in BSL2+.  Skilled staff provide help with instrument setup, data analysis, and consultation for experiment design. Training is available to enable investigators and their staff to run the analytical cytometer themselves.

Analytical Cytometry

Analytical Cytometry Instrumentation

The Becton Dickinson LSR II within the CVR is configured with four fixed alignment lasers and has the ability to detect up to 14 colors, utilizing a revolutionary optical design. The BD LSR II is configured with a 488 nm, 405 nm, 633 nm, and 355nm UV laser. At the heart of the BD LSR II are new digital electronics. The electronics digitize signals 10 million times per second in 16,384 discrete levels, expanding channel resolution and increasing detection sensitivity. A digital acquisition system allows for inter- and intra-beam compensation as well as the ability to threshold any parameter.

 

User Registration – Analytical cytometry

You must be trained in the instrument’s operation.  You can either travel to Becton Dickinson for training or you can be trained by Tim Sturgeon (sturgeon@pitt.edu, 412-383-5333), CVR Process Research Manager.  If you receive your training from a BD training center, the facility will require a copy of your user certificate prior to instrument usage. Contact Tim sturgeon for details on equipment reservation, billing procedures as well as current rates.


 

Analytical Flow Facility And User Responsibilities

As the CVR Process Research Manager, Tim Sturgeon is the manager for the flow facilities. His responsibilities include personnel training, machine maintenance, machine quality control, and assisting users in assay development and troubleshooting. In addition, Tim is responsible for ordering and stocking the supplies needed to keep the machines running. These supplies include QC and routine maintenance supplies but does not include tubes or other associated consumables.

The main responsibility of facility users is to prepare the machine for the next user at the completion of the session. These operations include:

1. Filling the sheath tank and emptying the waste tank at the end of your session.

2. Exporting your experimental data out of the FacsDiva browser to conserve hard drive space.

3.  Quick-cleaning the machine for the next user (15 minute process).

For more detail on use of the equipment please contact Tim Sturgeon (sturgeon@pitt.edu, 412-383-5333)

 

Analytical Flow Sample Preparation and Suitability

Samples to be run on the LSR II should have a volume of at least 300ul and be contained in a 12X75mm polystyrene tube (Falcon #352058). Samples should be fixed in 1-2% paraformaldehyde for at least 30 minutes prior to being run on the machine. In special circumstances, unfixed cells may be run on the machine.  Please discuss using unfixed samples with Tim Sturgeon prior to scheduled acquisition time. Samples that contain radiation should not be run on the LSR II.

The above information is a concise outline of the policies and procedures for operating the LSR II in The Center for Vaccine Research. Please feel free to discuss concerns or questions with Tim at any time.

 

Cell Sorting or FACS (Fluorescence Activated Cell Sorting)

FACS Instrumentation

The CVR has a BD FACSAria in a BIOProtecttII Class AII BSC in a BSL2+ facility as  well as one in our BSL3 facility, allowing for sterile sorting of infected or unifected cells of various BSL restrictions. The BD FACSAria instrument was the first benchtop sorter that incorporated a fixed-alignment cuvette flow cell that can detect up to 13 colors, for a total of 15 parameters. The cuvette flow cell allows for up to three air-cooled or solid state lasers at 488 nm, 633 nm, and 405 nm. Digital electronics allow for acquisition rates of up to 70,000 events per second. High-performance sorting is easy to set up, perform, and monitor with built-in BD FACS™ Accudrop technology and sort monitoring features within the software.

 
User Registration – FACS

Currently, the CVR runs both FACSAria facilities solely as manager-contracted sorting. For additional information on availability, use, scheduling or rates, please contact the CVR Process Research Manager, Tim Sturgeon (sturgeon@pitt.edu, 412-383-5333)


 

 

 

 

AutoMACS Pro

The features of MACS MicroBead Technology qualify the autoMACS® Pro Separator (Miltenyi)  as an alternative or complement to FACS and allow for a broad range of downstream applications such as flow cytometry , cell culture , functional assays, FISH, PCR and -omic approaches. The AutoMACS Pro performs automated magnetic cell sorting. Located within a BioProtect II biosafety cabinet in one of the CVR’s BSL2+ facilities sterile sorting of infected or uninfected samples is possible. The sample is purified over a column matrix that brings the field in proximity to the cells. The magnetically labeled cells are retained on the column, while unlabeled cells pass through and can be collected as the unlabeled fraction. The retained cells are eluted from the MACS Column after removal from the magnet.

For additional information on the cytometry equipment or its use, please contact CVR Process Research Manager, Tim Sturgeon (sturgeon@pitt.edu, 412-383-5333)

 

CTL IMMUNOSPOT

This is a CTL Immunopot elispot reader using Image Acquisition version 4.5 and Immunospot version 5.0 analysis software.  The reader is typically customized to provide fixed magnification for 96-well plates with excellent image quality, however is does offer additional plate formats including 24-, 48-, and 384-well capability. It is capable of performing basic and double-color counting.  

 

LUMINEX

A Bio-Rad Bio-Plex II suspension array system is available for use, both under BSL2/2+ or BSL3 conditions, by labs affiliated with the Center for Vaccine Research. The Bio-Plex II array system is a flow-based, dual laser system for identifying and quantifying up to 100 different analytes in a single biomolecular assay, using Luminex’s xMAP technology.  A variety of assays are readily available allowing for the study of cytokine/ chemokine levels, complex genetic interactions, as well as signal transduction pathways in a single small volume sample. A Bio-Plex Pro II array washer complements the array system allowing for the use of either magnetic or non-magnetic bead assays.

 

ABI 7900HT qPCR MACHINE

The ABI 7900HT fast real-time SDS system is a high-throughput real-time PCR system that detects and quantitates nucleic acid sequences. Continuous wavelength detection from 500-660 nm allows the use of multiple fluorophores in a single reaction. To induce fluorescence, the 7900HT system distributes light from an argon- ion laser excitation source to all sample wells. It then directs the resulting fluorescent emission through a spectro- graph to a charge-coupled device (CCD) camera.

Contact the CVR Process Research Manager, Timothy Sturgeon (sturgeon@pitt.edu, 412-383-5333)  for additional information and scheduling.

 

SURFACE PLASMON RESONANCE (SPR)

The CVR offers state-of-the-art SPR (Biacore 3000) instrumentation for label-free, real-time detection of biomolecular interactions and measurement of binding specificity, kinetic association and dissociation rates, binding affinity, and active concentration. The sensitivity and broad biomolecular sample compatibility of SPR also allows for the design of assays to assess more complex aspects of interactions such as binding thermodynamics, epitope mapping, binding mechanism resolution, and immunogen-induced antibody avidity responses in clinical sera samples. Our analyses are compatible with a wide range of biomolecules and can successfully measure interactions between proteins, peptides, small molecules, lipid membranes, carbohydrates, synthetic polymers, whole viruses, and whole cells.

 

SPR Sensorgram. A typical SPR experiment - (1) Ligand immobilization; (2)  analyte injection, complementary to the immobilized ligand results in population of the surface active sites leading to (3) saturation and a corresponding increase in the SPR response. (4) Replacing analyte injections with buffer will dissociate analyte from the surface, (5) until an injection of a regeneration buffer removes any remaining bound analyte to (6) regenerate the ligand-immobilized surface in preparation for the next analyte sample. From the SPR data the kinetics and affinity of the interaction and/or the active analyte concentration can be calculated.

 

The CVR currently offers two usage models for completion of SPR analyses:

Manager run - Researchers provide the samples of interest and all experimentation and analysis will be performed by the CVR.

Independent Use - Researchers will be trained in the independent operation. Once trained and approved for use, researchers will be free to reserve and operate the instrument independently with 24/7/365 availability.

For additional questions on access, use, or associated rates for the CVR SPR core,  please contact the CVR Director of Operations, Dr. Jodi Craigo (craigoj@pitt.edu, 412-624-4610).

HPLC

The CVR has a Waters’ Alliance HPLC system for routine chromatography needs.

Features and Benefits

  • Quaternary solvent blending supports isocratic and gradient applications with 11 unique gradient profile curves. Blend up to 4 solvents in any proportion with Auto-Blend Technology. Compatible with a wide range of solvent types to support reversed-phase, normal-phase, GPC, SEC, and IEX application.
  • Advanced solvent delivery capabilities. Serial flow path with pulse-free solvent delivery without the use of dampeners. Online solvent degassing and automated solvent compressibility for accurate flow. Integrated seal wash for robustness operation with all solvent types is standard.
  • Up to 120 vials housed in an optional, temperature-controlled environment.  Active and user programmable needle wash for low carryover.  Variable injection volumes from 0.1 to 100 uL standard, and up to 2 mL optional.  Supports STAT samples and auto-addition.
  • Detection capabilities include photodiode array, UV/Vis, fluorescence, refractive index, evaporative light scattering, electrochemical, conductivity, and mass detection.
  • Supported in Empower, MassLynx

 

Contact the CVR Process Research Manager, Timothy Sturgeon (sturgeon@pitt.edu, 412-383-5333)  for additional information and scheduling.

ADDITIONAL RESOURCES

The CVR has additional equipment including two fluorescent microscopes as well as other standard laboratory equipment. For question regarding availability please contact the CVR Director of Operations, Dr. Jodi Craigo (craigoj@pitt.edu, 412-624-4610).

Please see the Regional Biocontainment Laboratory-specific page for information on the resources available within the containment facilities of the RBL.