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    Molecular and Cellular Analytic Core

    NORC transparent black

    MCA CORE CONTACTS

    Director: Bryan Bergman, PhD
    Associate Director: Kartik Shankar, PhD
    Mitochondrial Sub Core: Matthew Jackman, PhD
    Lipidomics and GS/MS Analysis: Karin Zemski-Berry, PhD
    View the MCA Team

     

    ABOUT

    The overall aim of the Molecular and Cellular Analytical (MCA) core is to support the investigation of human and non-human metabolism from a cellular/molecular perspective. The core focuses on investigation in the following areas:

    Lipidomics and Mass Spectrometry

    GC/MS Analysis

    Mitochondrial Respiration

    Bioinformatic Analysis of RNAseq Data

    Lipidomics and Mass Spectometry

    Offering analytical services, as well as expertise in interpretation of lipidomic data, the Lipidomics/Mass Spectrometry services can quantitatively analyze the following lipids from plasma as well as cells and tissues (muscle, heart, liver, bone):

    • Triacylglycerols (TAGs) 
    • DAGs/Ceramides/Glycosphingolipids – diacylglycerols (DAGs), ceramides (Cer), dihydroceramides (dHCer), glucosyl ceramides (GluCer), galactosyl ceramides (GalCer), and lactosyl ceramides (LacCer)
    • Sphingolipids and Acyl Carnitines – sphingosine (SPH), acyl carnitines (AC), sphingomyelin (SPM)
    • Glycerophospholipids – includes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), and all lyso derivatives of these compounds
    • Eicosanoids – HETEs, HODEs, diHOMEs, diHETs (diHETrEs), leukotrienes, prostaglandins, thromboxane
    • Oxidized PCs – POVPC, PGPC, PONPC, and PAzPC

    Customer is responsible for sample homogenization and BCA assay to determine protein content for normalization purposes. The NORC Lipidomics Laboratory staff extracts the samples, runs the quantitative LC-MS/MS assays, and analyzes the lipidomic data.

    All samples need to be delivered or shipped in a -80C freezer box in the order they are listed on the Sample Inventory Form. Sample tubes MUST be numbered sequentially (1 to x, where is x is your last sample).  Any unique code for your samples can be recorded on the Sample Inventory Form in the sample description column.

    All academic analyses are subsidized by the Colorado NORC. Reduced pricing is available for junior investigators. Turnaround time is dependent on the number of samples submitted, the specific lipids of interest, and the number of samples in our queue.  For current pricing and to discuss sample analysis and turnaround time, please contact Karin Zemski-Berry. 

    Lipidomic Analysis Equipment

    ABSciex 3200 Acylcarnitines Sphingomyelins

    ABSciex 2000 TAGs

    ABSciex 2000 Trap DAG and Sphingolipids

    ABSciex 4000 Trap Eicosanoids

    ABSciex 5500 QTrap Deoxysphingolipids

    GC/MS Anlaysis

    The Bergman Lab has been performing GC/MS analysis for ongoing research as well as collaborative projects since 2004. The lab analyzes plasma samples for concentration and enrichment of glucose, glycerol, and free fatty acids (FFA). The methods used include Glucose and glycerol: Penta-acetate derivatization in EI mode and FFAs: PFB derivatives in CI mode. 

    All samples need to be delivered or shipped in a -80C freezer box in the order they are listed on the Sample Inventory Form.  Sample tubes MUST be numbered sequentially (1 to x, where is x is your last sample).  Any unique code for your samples can be recorded on the Sample Inventory Form in the sample description column. The NORC Lipidomics Laboratory staff processes the sample, runs the GC/MS assay and analyzes the data. 

    All academic analyses are subsidized by the Colorado NORC. Reduced pricing is available for junior investigators. Turnaround time is dependent on the number and types of samples to prepare and run, as well as the amount of samples in our queue. For current pricing and to discuss sample analysis and turnaround time, please contact Karin Zemski-Berry. 

    GC/MS Analysis Equipment

    Agilent 6890 GC/5973 MS

    Bioinformatic Analysis of RNAseq Data

    Next-Gen Sequencing. Our purpose is to provide existing members the NORC and researchers at the University of Colorado Anschutz Campus with a consolidated workflow for the management, processing, and biological interpretation of unbiased RNA sequencing gene expression data.

    Basics of the Service. We process high resolution RNA sequencing data (FastQ) and implement state-of-the-art computational and bioinformatics methods to supply the end user with statistically significant genes, annotated gene lists, canonical pathways, putative upstream regulators, disease states, gene interaction networks, and biological meaning specific to their research question.

    Mitochondrial Function Sub Core - Mitochondrial Respiration

    The Mitochondrial Function Sub Core of the MCA Core facilitates the comprehensive measurement of mitochondrial function and cellular bioenergetics for both academic and industry users by providing equipment for end users. Respirometry measurements may be performed on a host of biological samples including cultured/primary cells, permeabilized fibers or cells, and or isolated mitochondria.

    Training and professional development in the use and understanding of the various respiration methodologies is provided. Basic training/consultation is routinely provided for use of the Seahorse XF96 Extracellular Flux Analyzer, and the BioTek Cytation instruments. More in depth training/consultation is provided for the Oroboros O2k, and Strathkelvin systems.

    Mitochondrial Sub Core Equipment & Pricing

    Both a XFe96 Seahorse and a pair of O2k Respirometers are available for respirometry assays in intact cells, permeabilized cells, and isolated mitochondria. These assays can be used to quantify mitochondrial ATP production, maximal capacity of the respiratory chain, uncoupling, mitochondrial membrane potential, and ROS production. 

    Normalization of the XFe96 Seahorse data via cell number, genomic DNA, and total cellular protein, can be a time consuming processes. To facilitate a simple, time saving solution to normalization, the NORC MCA core has purchased a BioTek Cytation-1 Cell Imaging Multi-Mode Reader to work in concert with the XFe96 Seahorse for normalization purposes. Following a Seahorse assay run, Hoechst stain is added, and the Seahorse plate is then transferred to the Cytation-1 imager. Images and counts are then transferred back into the Wave software for normalization of the Seahorse ECAR and OCAR data. The entire normalization procedure takes approximately 30 minutes.

    Both a XFe96 Seahorse and a pair of O2k Respirometers are available for respirometry assays in intact cells, permeabilized cells, and isolated mitochondria. These assays can be used to quantify mitochondrial ATP production, maximal capacity of the respiratory chain, uncoupling, mitochondrial membrane potential, and ROS production. 

    Allows for the simultaneous monitoring of oxygen consumption together with either ROS production or mitochondrial membrane potential.

    Both a XFe96 Seahorse and a pair of O2k Respirometers are available for respirometry assays in intact cells, permeabilized cells, and isolated mitochondria. These assays can be used to quantify mitochondrial ATP production, maximal capacity of the respiratory chain, uncoupling, mitochondrial membrane potential, and ROS production. 

    Coupled to a 782 oxygen meter.

    The Jess is a fully automated capillary-based immunoassay method, requiring less sample, antibody, and time compared to traditional western blots. Simply pipette samples and reagents into the microplate, and the machine automatically separates proteins by size, manages antibody additions, incubations, washes, and detection. Up to 25 samples may be run in approximately 3hrs.  Multiplexing is also possible, as the Jess allows for 4 different ways to analyze proteins, Fluorescent (IR and NIR), chemiluminescent, and protein normalization.

    Current Pricing: 
    XFe96 Seahorse$119.76/run
    Cytation-1$25/run
    Oroboros O2k$142.50/O2k/day
    Protein Simple Jess$30/run

    In order to schedule/reserve the equipment above, you will need an iLabs account which can be set up here:  https://cu.corefacilities.org/account/login. 

     

    Once the iLabs account is obtained, please contact Matthew Jackman for access to the MCA core website reservations. 

    The following equipment is also available for study use on mitochondrial function if needed:

    High speed refrigerated floor centrifuge

    2 bench top centrifuges

    Microfuges

    Mettler semi-micro balances

    Dissecting microscope

    Colorado Nutrition Obesity Research Center (NORC)

    CU Anschutz Health and Wellness Center

    12348 East Montview Boulevard

    Aurora, CO 80045


    norc@cuanschutz.edu

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