Cell Sorting

Using a flow cytometer with cell sorting capabilities we can separate and purify cells or particles of interest for further analysis. Any cell or particle that can be made fluorescent can be separated and sorted into 96 well plates, tubes. Common types of samples include cells expressing a fluorescent protein, stem cells, tumor infiltrating lymphocytes, tumor cells, and white blood cell populations.

Immune Cell Phenotyping

Immunophenotyping is a common application of flow cytometry allowing you to simultaneously analyze multiple parameters on mixed populations of cells. Cells are stained with fluorochrome-conjugated antibodies targeted against antigens on the cell surface, or intracellular antigens following fixation and permeabilzation.

See references (1) and (2) for examples.

Immune Cell Function Analysis

Flow cytometry allows the sub-classification of lymphocytes on the basis of their immune function. Analysis of surface marker CD antigens can reveal imbalances or deficiencies in the various lymphocyte populations and thus can shed light on acquired or congenital immunodeficiencies.

Intracellular Cytokine Staining Analysis

Intracellular cytokine staining in combination with immunophenotyping allows you determine if particular cell subsets are activated by an experimental condition or drug. Cells are stimulated and incubated with a protein transport inhibitor such as Brefeldin A or Monensin so that secreted proteins are retained within the cells. Surface staining, followed by fixation and intracellular staining is then performed.

Intracellular Cytokine Staining Analysis

Intracellular cytokine staining in combination with immunophenotyping allows you determine if particular cell subsets are activated by an experimental condition or drug. Cells are stimulated and incubated with a protein transport inhibitor such as Brefeldin A or Monensin so that secreted proteins are retained within the cells. Surface staining, followed by fixation and intracellular staining is then performed.

Receptor Occupancy Analysis

Receptor occupancy can provide insight into the pharmacodynamics of a drug or protein. We can titrate your test article on cells expressing a particular receptor and stain with antibodies that are specific for the unoccupied receptor. Analysis of MFI or percent positive cells provides a dose-response curve indicating the concentration required to reach maximum receptor occupancy.

Gene Therapy

Gene replacement therapy holds promise for correcting deleterious genetic mutations that result in a loss of function. Flow cytometry can be used to see if a synthetic gene of interest is expressed using antibody staining for the protein of interest, epitope tag or a co-expressed fluorescent protein.

Signal Transduction/Cell Signaling

Signal transduction can be analyzed by flow cytometry with antibodies targeting phospho-epitopes involved in a signaling pathway of interest. (3)(4)

Apoptosis Pathways Analysis

During apoptosis, cells release blebs from the cell membrane. When this occurs, Annexin V transitions from the intracellular side of the cell membrane to the extracellular side. Co-staining for Annexin V and 7AAD can be used to assay whether cells are alive, entering apoptosis or dead.

Cytotoxicity Assays

We offer several types of cytotoxicity assays. In our FACS cytotoxicity assay, cells of interest are labelled with a covalently binding dye and incubated with the drug, protein or cell-based test article. Following incubation, cells are stained with a fixable Live/Dead dye or 7AAD. FACS analysis allows for easy identification of cells of interest and assessment of cell death. Other cytotoxicity assays available include impedance-based real time cytotoxicity assay on the xCelligence RTCA instrument, LDH release and luminescence-based killing assays.

Cell Cycle Regulation Analysis

Cell cycle analysis can be performed using DNA-specific stains. As cells progress through the cycle, DNA content increases prior to cell division. This can be a useful readout when studying drugs to assess toxicity or therapeutic effect in the case of anti-neoplastic drugs. Flow cytometry also allows for easy interrogation of additional phenotypic markers as needed.

Cell Enumeration

Absolute cell enumeration can be performed by including counting beads and an initial volume measurement in your flow cytometry experiment. Bead events are easily identified by a unique combination of high SSC and bright fluorescence in multiple channels.

Tumor Suppressor Gene/Protein Expression

ProMab can look for protein expression by flow cytometry. Flow cytometry can easily be added to in vitro and in vivo studies to comprehensively profile changes in immune cell populations in response to therapeutics. With a streamlined process for tissue dissociation, cell isolation, staining and acquisition, we can process hundreds of blood or tissue samples each day, giving clients the benefit of actionable data in near-real time.

Proliferation and Cell Cycle Analysis

Several FACS-based methods are available to measure cell proliferation such as dye-dilution (CFSE) or staining for proliferation related antigens (Ki67, PCNA). >>> Learn more

Apoptotic Checkpoints

Classical apoptotic checkpoints can be rapidly examined by flow cytometry.

Cell cycle analysis assays consist of staining DNA with DNA binding dye, fixing cells with a 70% ethanol solution which permeabilizes the cells and then staining cells with the dye of your choice.

Some dyes can enter living cells and stain DNA without damaging the cells (e.g. Hoescht 33342), and samples can be acquired at a low flow rate with linear amplification to determine the cell cycle phases. (5)

Antigen-Specific Cell Responses

To analyze antigen specific responses— cells are stimulated with a specific antigen later and analyzed for a functional readout such as cytokine production, proliferation, cell activation, conversion to effector or memory cell type. Analysis of the proportion of antigen specific T cells can be accomplished with peptide-MHC tetramer staining. (6)