In Vitro Oncology

Our in vitro services now offers the sandwich enzyme-linked immunospot (ELISpot or FluoroSpot) assay to measure the frequency of cytokine secreting cells in cell suspensions.  This assay allows the secreted cytokine to bind to the coated antibody resulting in a single spot. The CTL S6 Universal analyzer is used to capture quality images and analyze the spots.  Equipped with four optical filters, the CTL analyzer is capable of detecting up to three analytes simultaneously using either colorimetric or fluorometric readouts.  ELISpot or FluoroSpot kits are compatible with blood, lymphoid, and solid tissue.  The samples are routinely performed in duplicate or triplicate to obtain an average to quantify the amount of spots in each sample.

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

Immunohistochemistry and Validated Markers

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.

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.

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.

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.

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.

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.

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.

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.

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 Covance'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.

Read our related blog article regarding luciferase

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.

• Albumin
• Alkaline phosphatase
• Alanine aminotransferase
• Aspartate aminotransferase
• Blood urea nitrogen
• Calcium
• Cholesterol

• Albumin
• Alkaline phosphatase
• Alanine aminotransferase
• Aspartate aminotransferase
• Blood urea nitrogen
• Calcium
• Cholesterol