Our in vitro services offer a full suite of molecular and cellular biology assays to address the invaluable need for analysis of target and drug activity before, during, and after the in vivo experiment. Our expert in vitro scientists can perform off-the-shelf tests as well as work closely with our clients to develop a custom assay to suit their specific program’s needs.
And you can also partner with our team of flow cytometry experts who have extensive experience designing and validating experiments, processing multiple types of samples, and acquiring and analyzing data. We offer basic to comprehensive immune profiling using one of our standard immunophenotyping panels, phospho-flow, functional assays, and custom antibody panels and consultation to meet your unique needs.
Our In Vitro Services Include:
Protein Expression Analysis
We have a Synergy 2 plate reader with a red-shifted PMT, a Cytation 3 plate reader containing monochromators that can measure a wide range of wavelengths, and a 405LS Microplate Washer to reduce incomplete or uneven washing. We have extensive experience using ELISAs from multiple vendors and with multiple sample types such as cultured cells, whole blood, fresh tissues, and frozen tissues. Absorbance-based or fluorescence-based ELISA kits can be used to produce a standard curve and accurately quantify the amount of a specific protein in your samples. To obtain the most accurate results, we routinely generate standard curves from protein standards in duplicate or triplicate and quantitates your protein of interest in each sample from triplicate wells.
In collaboration with our clients we have performed many successful Western blots against a large number of oncology relevant targets. The staff has extensive experience performing Western blots from multiple sample types such as cultured cells, fresh tissues, and frozen tissues. With the Typhoon Imaging System, chemiluminescent, chemifluorescent, and fluorescent secondary antibodies can be detected allowing for the identification of multiple proteins of interest at one time. We also have the ability to quantify the bands on a membrane with ImageQuant software to determine the size and relative intensity of each band for each sample.
We have two Attune NxT flow cytometers with four lasers capable of detecting fluorescent compounds in 14 channels. Whether you need to examine the abundance of a specific protein or multiple proteins on the surface or inside cells, our flow cytometry experts together with the Attune NxT can make it happen. Monitor the abundance or localization to/from the plasma membrane of a specific protein over time with treatment or compare the levels of a protein in different cell lines or mouse strains. Combine the detection of a specific protein with any of our other common flow cytometry cell process assays, (below) such as the apoptosis, cell cycle, or cell viability assay to determine if the abundance of the protein changes at different stages of a cell process. We have successfully detected hundreds of different proteins in samples such as cultured cells, blood, tissues, and organs. In blood, we have successfully detected proteins using a no-wash no-lyse assay which reduces the amount of manipulation and allows the analysis of very small volumes of blood. Find out more about our Flow Cytometry Services.
Cytometric bead arrays
We also have a Luminex® 100™ System. This system is a flexible analyzer based on the principles of flow cytometry. The system enables you to multiplex (simultaneously measure) up to 100 analytes in a single microplate well, using very small sample sizes. The system delivers fast and cost-effective bioassay results on many assay formats including nucleic acid assays, receptor-ligand assays, immunoassays, and enzymatic assays. Commercial, pre-made, multiplexed plates are available to measure the components of entire cell signaling pathways or cytokines such as the AKT pathway, apoptosis, chemokines, cytokines, inflammatory cytokines, growth factors, and the TH1/TH2 pathways.
Analyze your tissue samples with IHC staining to detect abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. We offer a full suite of in-house histology and IHC services including custom work, and will be adding markers routinely. Our services include:
- Panels: Cancer, Immuno-Oncology
- Normal Tissue, Tumor, Xenograft, Cell Line, Validation, Immunofluorescence, Immunocytochemistry, Multiplex
We have an Olympus CKX53F inverted fluorescence microscope to view fluorescent compounds/proteins in live cells and a BioTek Cytation 3 imaging multi-mode reader to view fluorescent compounds/proteins in live or fixed cells. With the use of fluorescence microscopy on live cells, we can quickly determine the percentage of cells containing a fluorescent compound or protein without manipulating the cells. With fixed cells, we can determine the cellular localization of specific proteins within the cells after staining with fluorescently-labeled antibodies and using fluorescent dyes such as DAPI, FM 1-43, Calcein, or DiOC6 to define specific compartments within a cell.
DNA / RNA Expression Analysis
We use a StepOnePlus™ Real-Time PCR System that can read four different colors in a 96-well plate and supports assays such as SNP genotyping, gene expression analysis, microRNA expression, translocation analysis, and gene detection. Our PCR specialists can help you to determine the expression of specific genes in cultured cells, fresh tissues, or frozen tissues. Whether you need to measure the expression of genes in different cell lines, strains of mice, tissues, or tumors, or monitor the expression of different genes with different treatments, our PCR specialists can help you design a study that’s right for you.
Cell line STR profiling
Genetic instability of some cell lines can result in changes at the DNA level that can compromise research results. Cell line authentication via STR (short tandem repeat) analysis will ensure that the research is being conducted on the correct cells, maximizing reliable results, supporting publication efforts, and reducing the risk of wasted time and resources. Authenticate your cell lines by comparing the STR results to those in published sources. The STR profile of newly generated cell lines should be established so that the STR profile of future passages can be compared and the stability of the DNA can be monitored. We collaborate with a partner laboratory that specializes in STR profiling techniques and analysis.
Cell line and tumor gene expression profiling
Gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once to create a global picture of cellular function. The RNA is sequenced using next-generation sequencing, quantified, and aligned to thousands of known RNA sequences from specific genes. These profiles can, for example, distinguish between cells that are actively dividing, show how the cells react to a particular treatment, or determine which genes are differentially expressed in different cell lines, strains of mice, tissues, or tumors. Covance collaborates with a partner laboratory that specializes in sequencing the RNA. Once we obtain the sequencing information, we have the software to quantify, align, and analyze the data. We can compare the expression of single genes, sets of related genes, groups of pathway-specific or process-specific genes, and entire genomes between samples and display the comparative results in straightforward graphs and figures.
Apoptosis via flow cytometry
Apoptosis is a normal physiologic process, the program of which is characterized by certain morphologic features, including loss of plasma membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. FITC Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with FITC Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) to identify early apoptotic cells (PI negative, FITC Annexin V positive). Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Let us determine if treatment with your compound includes apoptosis in your samples and receive data regarding the percentage of cells in each sample that are alive, apoptotic, and dead. Our flow cytometry team can perform apoptosis assays on multiple types of samples such as cultured cells, blood, or tissues, and, if desired, analyze specific subsets of cells within each sample.
Cell cycle via flow cytometry
When staining the DNA of a cell with a fluorescent DNA binding agent, such as propidium iodide (PI), the relative amount of DNA in the cell can be measured via flow cytometry and therefore, the stage of the cell cycle can be elucidated. During G0 or G1, a cell contains 46 chromatids; during the S phase, the chromosomes duplicate, and by the end of S phase, G2, or M phase, there are 92 chromatids. The effects of cell cycle checkpoint inhibitors can be confirmed using this method. Our flow cytometry experts are able to confirm the effects of cell cycle checkpoint inhibitors with the method mentioned above, as well as determine the percentage of cells in different stages of the cell cycle in samples such as cultured cells, blood, and tissues.
Cell viability, proliferation via flow cytometry
Ubiquitous intracellular esterase activity and an intact plasma membrane are distinguishing characteristics of live cells. A simple viability assay routinely performed using an amine reactive fluorescent dye that is non-permeant to live cells, but permeant to the cells with compromised membranes. Thus, it can be used to assess live vs. dead status of mammalian cells via flow cytometry. These assays are more sensitive than Trypan blue exclusion, a commonly used method for live/dead cell discrimination. Monitor the viability of cells in your samples over time with treatment or differing procedures. Combine the cell viability assay with other flow cytometry assays such as the cell cycle assay or specific protein abundance analysis to exclude the analysis of dead cells. The flow cytometry team also has experience measuring the proliferation of T-cells in mixed lymphocyte reactions using CFSE staining.
Intracellular signaling via phospho-flow cytometry
We also offer phospho-flow cytometry, which is a valuable tool for screening new drugs for their effects on the activation of cell signaling pathways in vitro; as well as having the ability to measure drug effects in vivo by multiplexing the technique with immunophenotyping cell surface markers to distinguish analysis in different cell subsets. This platform uses fluorescence-labeled antibodies that recognize proteins only when phosphorylated on specific amino acid residues that regulate their function. Phospho-flow data is highly consistent and reproducible, which in turn creates a unique platform for cell signaling analysis. For more information, read our blog related to this topic: “Phospho-Flow Cytometry for the Screening of Intracellular Signaling Events in Cancer Cells and Immune Cells” or view our webinar on this subject matter.
Cell and Tissue Processing
Cell sorting using flow cytometry is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Cell sorting can separate live cells based on any scatter, fluorescent signals, or combination of both. We have experience separating live vs. dead cells, cells expressing a specific fluorescent protein, or cells expressing a specific surface marker or set of surface markers. After separation, cells can be cultured and/or used in any downstream assay. We have also successfully created multiple single cell clones expressing different levels of specific proteins from multiple cell lines. We collaborate with a partner laboratory that has multiple cell sorting flow cytometers and years of experience sorting cells in multiple ways.
Automated tissue processing to viable cells
Covance can dissociate tissues into single-cell suspensions by combining mechanical dissociation with enzymatic degradation of the extracellular matrix, which maintains the structural integrity of tissues. The Miltenyi Biotech Dissociation Kit in conjunction with the gentleMACS™ Octo Dissociator, produces gentle, rapid, and effective generation of single-cell suspensions from primary human or mouse tumor tissue or xenografts. The assay has been optimized for a high yield of tumor cells and tumor infiltrating lymphocytes (TILs), while preserving cell surface epitopes. Dissociated cells can be subsequently cultured, stained for internal/external protein markers, isolated using cell sorting, or used in many other downstream assays. Other than tumor tissues, the flow cytometry experts have many years of experience dissociating many different tissues such as spleens, lymph nodes, lungs, and skin.
Automated tissue processing to protein or DNA/RNA homogenates
Covance can provide frozen protein homogenates containing a battery of protease and phosphatase inhibitors or DNA/RNA homogenates in TRIzol® or RNAlater®. We have a BeadBlaster™ 24 Microtube Homogenizer that completely lyses, grinds, and homogenizes a broad variety of biological samples from soft tissue to bone. Even the most difficult and resistant samples are fully homogenized. The stainless steel tube carrier subjects up to 24 samples to an optimized, high speed, 3D motion producing high energy impacts between the samples and micro-beads to release cellular contents. After homogenization, samples can be centrifuged and the supernatant collected for further processing.
Covance can grow and expand your cell lines for implantation into hundreds of mice or freeze as many vials as necessary at the cell concentration you need. We have fourteen CO2 incubators, three full-sized biosafety cabinets, two cell automated cell counters, and a vast amount of experience growing hundreds of different cell lines in dishes, flasks, roller bottles, spinner flasks, and multi-layered flasks.
Covance can monitor the cell morphology of your cells in culture using an Olympus CKX53F inverted fluorescence microscope with phase contrast and a 1.4MP CCD camera. View pictures of your cells before, during, and after treatment, over extended periods of time, or at specific time points before implantation to verify that the cells are growing normally.
We can monitor the growth of your cells over time and compare different cell variants, cells grown in different media, or cells grown with different treatments. We have two Luna Automated Cell Counters that can accurately distinguish the live from the dead cells and count each population. Our cell culture team has a vast amount of experience culturing multiple types of cells including tumor cells, primary cells, and neurospheres. The team also has experience performing colony formation assays to detect the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells in vitro. Our flow cytometry team has experience monitoring the proliferation of stimulated T-cells or T-cells in mixed lymphocyte reactions using CFSE staining.
High-content image analysis
Covance has the ability to stain cells and acquire fluorescent images using the Cytation 3 Imaging Plate Reader. Once the images have been captured, we have specialized software that can count nuclei, count live vs dead cells, count stained cells as positive vs. negative, and measure the size, circularity, area, perimeter, and intensity of each color in the images.
Image-enabled cell line generation
Firefly (P. pyralis) luciferase is an enzyme that produces bioluminescence in the presence of D-luciferin. The DNA for the luciferase 2 (luc2) gene, along with a puromycin resistance gene, is incorporated into the genome of the cells using a lentiviral transduction system. While culturing the cells, puromycin is added to the medium to select only the cells with the inserted luciferase gene. Once the luc-enabled cells are injected into a mouse or rat and D-luciferin is administered, the light produced can be detected with MI Bioresearch’s IVIS® Spectrum in vivo imaging system. The in vitro services team has experience performing every step of the process from stably transducing cells, to selecting the transduced cells, to measuring the light output of the cells.
Cytotoxicity/proliferation assays are widely used to screen for cytotoxicity in compound libraries. If you are interested in developing a therapeutic agent that targets rapidly dividing cancer cells, we can test your cytotoxic compounds. We can also screen “hits” from initial high-throughput drug screens for unwanted cytotoxic effects before you invest in your agent as a pharmaceutical. MI Bioresearch can measure the toxicity of your compound in your choice of over 400 cell lines that we have available. We provide graphs and IC50 values for each cell line and compound.
Blood and Plasma Analysis
Complete blood counts*
We partner with a laboratory that specializes in the determination of the amount of each major component in blood samples from your study using a complete blood count (CBC).
A complete blood count test measures several components and features of blood, including:
- Red blood cells
- White blood cells
- Mean corpuscular volume
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
Abnormal increases or decreases in cell counts as revealed in a complete blood count may indicate that the animal has an underlying medical condition.
Blood chemistry tests or panels are groups of tests that measure many chemical substances in the blood that are released from body tissues or are produced during the breakdown (metabolism) of certain substances. A blood chemistry screen provides information about the general health of an animal, helps look for certain problems, and finds out whether treatment for a specific problem is working. We collaborate with a partner laboratory that specializes in the testing of blood for individual components or panels of components such as the full chemistry panel that includes:
- Alkaline phosphatase
- Alanine aminotransferase
- Aspartate aminotransferase
- Blood urea nitrogen
- Creatine kinase
- Total bilirubin
- Total protein
- Electrolytes (Na, K, Cl)
Pharmacokinetics, sometimes abbreviated as PK, is a branch of pharmacology dedicated to determining the fate of substances administered externally to a living organism. The substances of interest include pharmaceutical agents, hormones, nutrients, and toxins. It attempts to discover the fate of a substance from the moment that it is administered up to the point at which it is completely eliminated from the body. Pharmacokinetics describes how the body affects a specific substance after administration through the mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body (e.g., by metabolic enzymes such as cytochrome P450 or glucuronosyltransferase enzymes), and the effects and routes of excretion of the metabolites of the substance. Pharmacokinetic properties of substances may be affected by elements such as the site of administration and the dose of administered substance. These may affect the absorption rate of the substance. We collaborate with a partner laboratory that specializes in the detection of your agent in specific tissues.
*Denotes services performed at validated partner labs
Other standard techniques are available. Custom assay development is also available to meet our clients’ individual needs.
In Vitro Equipment
Acoustic focusing flow cytometer 4 lasers, 16 parameters
Microplate sample processor and pipetting system
Imaging system with blue laser module
Multi-modality plate reader
High content imaging system
Luminex® 100™ System
Analyte Multiplexing System
Inverted fluorescence microscope with 1.4MP CCD camera
Real-time PCR system
Large-scale Cell Culture Production
Production facility with 14 incubators and three biosafety cabinets