BloodCenter of Wisconsin

BloodCenter of Wisconsin

LocationLocationNameDescriptionLinkCTSI Core/ServiceFree Core Access
LocationLocationNameDescriptionLink
BloodCenter of WisconsinbcwBIOPHYSICS CORE More InfoThe Biophysics Core consists of one system, the BIACore 3000.

The Biophysics Core at BloodCenter of Wisconsin’s Blood Research Institute provides the data gathering services of this system and the dedicated computers to all its investigators and outside users as capacity permits.

The BIACore 3000 uses the optical phenomenon of surface plasmon resonance (SPR) to monitor the formation and dissociation of bio-molecular complexes on a sensor surface as the interaction occurs. SPR is a non-invasive technique, which measures the mass concentration of biomolecules in close proximity to a specially prepared surface. By covalently attaching one molecule (the ligand) to the surface of a chip that forms one wall of a flow cell, then exposing it, under the conditions of continuous flow, to a second molecule (the analyte) in solution, interactive measurements can be made that are essentially independent of the nature of the biomolecule without the labeling of the interactants.
NoNo
BloodCenter of WisconsinbcwFLOW CYTOMETRY CORE More InfoThe Flow Cytometry Core contains 4 Becton Dickinson cytometer systems.

Instrumentation and Applications
BD LSRII Flow Cytometer
Capable of 10 color and 12 parameter acquisition
4 laser system includes blue 488nm, red 640nm, yellow-green 561nm and UV 355nm
BD HTS can be used on this instrument. Capable of reading 96 well and 384 well microtiter plates

BD LSRII Special Order System
Capable of 12 color and 14 parameter acquisition
4 laser system includes blue 488nm, red 640nm, green 532nm and violet 405nm
BD HTS can be used on this instrument. Capable of reading 96 well and 384 well microtiter plates
BD FACSAria Cell Sorter
Capable of 10 color and 12 parameter acquisition
4 laser system includes blue 488nm, red 633nm, green 532nm and violet 407nm

Accuri C6
Capable of 4 color and 6 parameter acquisition
2 laser system includes blue 488nm and red 633nm

The core provides data analysis with the following software:
Analysis workstations are available as well as training for flow core users.

FlowJo
FCS Express
FACSDiva
CFlow Plus

Miltenyi Biotec Magnetic Cell Separation Workstation
This workstation consists of a stand and 4 separation magnets that can be reserved for use. The usually vast initial cell population can be much reduced and greatly enriched for the target cell, with little or no added abuse to the cells. This cuts sorting time and usually increases viability.
NoNo
BloodCenter of WisconsinbcwHISTOLOGY CORE More InfoThe Histology Core provides services for tissue sectioning, preparation and staining. This core is operated jointly with the Physiology Department of the Medical College of Wisconsin where it is physically located. Core staff provide services in tissue processing, embedding, sectioning, and basic staining procedures. A cryostat is available for frozen section preparation.

Instrumentation and application
Automated tissue processor
This processor is used for paraffin sections. Tissues are preferred in 10% neutral buffered formalin.

Sakura automated stainer
The stainer can process 40 slides per run, hematoxylin and eosin (H & E). Dewaxing and hydrating services are available. These are useful when the user’s lab wants to perform its own specialized staining procedures.

Cryostat
The cryostat is used for frozen sections. OCT mounting media is available.

Manual staining
Stains available include trichrome, iron, periodic acid schiff (PAS) and others.
NoNo
BloodCenter of WisconsinbcwHYBRIDOMA CORE LAB More InfoThe Hybridoma Core Lab provides services and products related to the production of monoclonal antibodies. Basic procedures include mouse immunization, with immunogen provided by requestor. Subsequent boost and bleeds are done to check antibody titer in the animal. Spleen cells are fused with a non immunoglobulin-secreting mouse myeloma cell line.

The hybridomas are cultured in standard selection medium. These hybridomas are screened by a variety of methods by the requesting lab, or by the Hybridoma Core by ELISA. Up to 10 are cloned by limiting dilution and screened again. Once a stable cell line is produced and its isotype is determined, the core lab can produce ascites in vivo, or use in vitro methods of antibody production.

Cells are frozen at each stage of production and are kept in two locations. Generally, fusions are done with Balb/c mice. The Hybridoma Core lab offers related services as time and demand permits.

The Hybridoma Core also provides other tissue culture services. Endothelial cells from human umbilical vein are available upon request. Secondary passage and higher are kept in stock and can be ready with a week’s notice. Primary cultures require more advanced notice.

Core Policies and Services
Ascites production comes from in-house hybridoma or ATCC cell lines. If there is a sufficient supply, 3-15 ml of ascites can be provided from freezer inventory within 24 hours. If the ascites need to be produced in mice or in vitro, the wait time can be 3 weeks to 3 months depending on demand.

New fusions are on a first come, first served basis. Preference is given to BRI researchers. Generally, the requesting lab needs to provide about 1 mg of purified protein or peptide coupled to a carrier protein. The immunization period is typically 3 to 4 months. Screening, cloning, subcloning and freezing of cells can take an additional 3 to 6 months. All screening methods are to be worked out prior to fusion date. A technologist and the Scientific Director meet with each requesting lab to discuss the process and screening approach.
NoNo
BloodCenter of WisconsinbcwIMAGING CORE More InfoThe Imaging Core at BloodCenter’s Blood Research Institute (BRI) provides services in microscopy and analysis of two-dimensional images. Staff assists investigators and technologists through training for the use of microscopes and software packages that digitize images.

Instrumentation and Applications
Zeiss Lumar .V12, Dissecting scope
The Lumar is used for specimen dissection and analysis. It uses a 1.6X or 0.8X objective allowing for high resolution of specimens and great working distance. Fluorescence filter sets allow for the excitation of GFP, CFP and Rhodamine stained samples. Images can be acquired using a 5 megapixel Zeiss AxioCam MRc 5 camera. Perform further image analysis using Zeiss AxioVision 4.5 software.

NikonTE200 with Hoffman modulation, Inverted
Used for live cell cultures and prepared mounted slides. This microscope is outfitted with PLAN Fluor objectives 2X, 4X, 10X, 20X, 40X, and 60X. Fluorescence images can be acquired using filter sets for Hoeschst, DAPI, FITC, Texas Red, and Cy5. Images can be taken with either a Diagnostic Instruments Spot RT color camera using spot advanced software or a Photometrics CoolSNAP ES camera using Metamorph 6.1 software.

Nikon E600, Upright
Used for analysis of prepared mounted slides. This microscope is outfitted with Plan Fluor objectives ranging from 4X -100X. Images can be acquired using a Diagnostic Instruments Spot insight color camera using spot advanced software.

Zeiss AxioScope, Upright
Used for analysis of prepared mounted slides. This microscope uses high resolution Plan Neofluar optics ranging from 10X -100X. Fluorescence images can be acquired with filters sets exciting for DAPI, FITC Tex Red, Dual, and Rhodamine. Acquire images with a Photometric SenSys camera using Metamorph 6.1 software.

Metamorph imaging software
This software is used to measure and quantify different aspects of images. For example, sizes and numbers of cells, circumference of structures, and areas of portions of interest can be determined using this high throughput system.
NoNo
BloodCenter of WisconsinbcwMOLECULAR BIOLOGY CORE More InfoThe Molecular Biology Core Laboratory offers automated DNA sequencing services. In addition, the Core houses and maintains a real-time PCR detection system, and a suspension array system.

Instrumentation and Applications
ABI 3100×1 Genetic Analyzer
Automated sequencing is performed on an ABI 3100×1 Genetic Analyzer. This capillary-based platform allows for processing of up to 96 samples in an 18-hours. Samples are run within 1 to 2 days and yield a high-quality read length of up to 600+ base pairs.

Applied Biosystems 7500 Real-Time PCR System
This 7500 RT PCR System uses a 5-color platform calibrated for FAM/SYBR Green I, VIC/JOE, NED/TAMRA/Cy3, ROX/Texas Red, and Cy5 dyes.

Bio-Plex Array Reader and Microplate Platform
The flow-based, dual-laser array reader classifies each bead and its associated assay, and quantitates the amount of analyte captured. The microplate platform permits automated processing of samples from 96-well microplates in approximately 30 minutes. Using as little as 12 µl of serum or other biological sample per multiplex assay, this bioassay system allows for simultaneous detection and quantitation of up to 100 different analytes in a single well.

DNA Sequencing Services
Generally, users perform their own cycle sequencing reactions using ABI’s Big Dye Terminator chemistry (BRI internal users can purchase their sequencing reagents from the Core). The turnaround time is typically 1 to 2 working days after sample submission. For a nominal fee, users may request complete sample sequencing reaction preparation. Templates and primers are submitted separately in water. Users who opt for this service should contact us prior to sample submission to ensure proper processing of your samples.

Quantitative (real time) PCR Policies
Interested users should sign up before using the instrument. Sign-up is done on Outlook Public Folders under Core Schedules folder.
NoNo
BloodCenter of WisconsinbcwPROTEIN CHEMISTRY CORE More InfoThe Protein Chemistry Core Lab of the Blood Research Institute was established in 1988 and offers custom peptide synthesis and purification utilizing both natural and non-natural amino acids. Modifications such as biotinylation, fluorescent labeling, conjugation to carrier proteins, cyclization and incorporation of stable isotopes are routinely done. In addition, the Core Lab now supports isoelectric focusing, 2-D gel electrophoresis and digital gel imaging. The Core Lab has been a member of the Association for Biomolecular Resource Facilities since 1995.

Instrumentation and Applications
Peptide synthesis
The CEM LIBERTY1 is a microwave-assisted single channel instrument with UV monitor that generates high purity peptides with fast cycle times. The synthesis scales are from 0.05mmol to 3mmol. Peptides are synthesized using FMOC chemistry on solid supports or resins. Many modifications are possible including N and C terminal labeling or capping, side chain modifications, backbone modifications and stable isotope labeling. Peptides are purified by reverse-phase HPLC on a Beckman System Gold with a UV detector. Peptide masses are verified by MALDI-TOF mass spectral analysis. Synthetic peptides are delivered as dry lyophilized powders. Yield is based on scale and sequence.

Useful Links
Technical Resource Library for Peptide Synthesis Sigma-Aldrich
Peptide and Organic Synthesis Technical Resources EMD Millipore

Isoelectric Focusing / 2-D Gel Electrophoresis / Electroblotting
The technique of two-dimensional electrophoresis involves separating proteins in the first dimension according to charge (isoelectric focusing), followed by MW separation in the second dimension by SDS-PAGE. Isoelectric focusing is performed using a Biorad Protean IEF cell. This system uses immobile pH gradients (IPG) gels adhered to a plastic back.

The proteins are then visualized by staining the gel with Coomassie stain, silver stain or fluorescent dyes. This two dimensional array will produce spots that correspond to single protein species in the sample. Using this technique, different proteins can be separated and information such as pI, molecular weight and protein abundance can be determined. 2D-gels can be electroblotted to PVDF or nitrocellulose membranes for further analysis.

Digital Imaging
The Licor Odyssey Imaging system uses direct infrared detection that provides accurate quantification, sensitivity and a wide linear range. Two IR channels can be read simultaneously to probe two separate targets or to increase quantification accuracy by using the second channel for normalization. The Odyssesy is suitable for gels, membranes and glass slides.

The GE Healthcare Typhoon Trio is a variable mode imager that can be used for the acquisition of fluorescent or chemiluminescent data. The scanner is capable of imaging gel sandwiches, agarose and polyacrylamide gels, membranes, microplates and microarrays. Multiple dyes can be analyzed per sample. After acquisition, the images can be quantitated using ImageQuant TL from GE.

Core Policies and Services
The average turnaround time for standard peptide synthesis is two weeks, longer if modification or non standard amino acids are required. The peptide yield depends on the length and the purity required. The user will be advised if the project does not seem feasible or if low yields are expected.

Training is required before operating the Protean IEF, the LICOR Odyssey or the Typhoon Trio. Online calendars are maintained for the Protean IEF, Typhoon, Odyssey. Paper calendars are maintained for the AKTA and the Agilent. Users must sign up in advance to use these instruments.
NoNo
BloodCenter of WisconsinbcwVIRAL VECTOR CORE More InfoThe Viral Vector Core is shared between the Blood Research Institute and the Medical College of Wisconsin. It is located in the east wing of the BRI, room 2025. Vector systems used by the core include those based on lentivirus, retrovirus, adenovirus, and adeno-associated virus. This core provides services in the areas of vector design, gene silencing and protein expression, including construction, amplification, purification and titration. Additional services include cloning, mutagenesis, and plasmid DNA preparation.

LOCATION

Blood Research Institute, 8733 Watertown Plank Road

SERVICES

Lentiviral/Retroviral Vector Production:
Small-scale (plates) and large-scale (roller bottles)

Lentiviral/Retroviral Vector Titration:
Flow cytometry assay, integration qPCR, replication competence testing

Adenoviral Vector Production:
Recombination and transfection, small and large-scale amplification, purification

Adenoviral Vector Titration:
Flow cytometry based antibody assay

Adeno-associated Viral Vector Production:
Small and large-scale production and purification

Other:
Vector construction, site-directed mutagenesis, plasmid prep

*Various viral vectors expressing specific reporter genes such as GFP, YFP, mCherry and Cerulean can be purchased for testing and generation of preliminary data
NoNo

NIH Funding Acknowledgment: Important Reminder – Please acknowledge the NIH when publishing papers, patents, projects, and presentations resulting from the use of CTSI resources by including the NIH Funding Acknowledgement.

PARTNERS

Zablocki VA Medical CenterMedical College of WisconsinMSOE