We offer standardized immunoassays for human and mouse cytokines/chemokines which can be used to assay serum, plasma, cell culture supernatants, tissue homogenates, or other fluids. We measure multiple cytokines/chemokines simultaneously using Luminex bead-based kits. Our standard panels include 62 analytes for human and 39 for mouse. Custom panels are also available for projects of sufficient size.
Flow cytometry is used to assess immune cell frequency, phenotypes and functions. We offer standardized immunophenotyping, phosphoepitope, and intracellular cytokine assays for human blood cells (up to 18 color). Our assays have all been optimized for use with cryopreserved PBMC; use with fresh samples must be coordinated in advance.
Mass cytometry, or CyTOF (Fluidigm), is a variation of flow cytometry in which antibodies are labeled with heavy metal ion tags rather than fluorochromes. Readout is by time-of-flight mass spectrometry. This allows for the combination of many more antibody specificities in a single sample, without significant spillover between channels. We have standardized immunophenotyping, intracellular cytokine, and phosphoepitope CyTOF panels with 32-38 antibodies each.
B, T and APC Functional Studies
We have developed assays to identify, clone and expand beta (b) cell antigen (Ag) specific B and T cells from blood of patients with or at-risk for type 1 diabetes (T1D). In combination with the above technologies, this allows for the identification of B and T cells specific for b cell Ags and determination of their function (e.g. activation marker or cyotokine/chemokine expression). We also have developed assays to identify, sort and probe the function (e.g. Ag presentation, cytokine/chemokine secretion) of Antigen Presenting Cell (APC) subsets, including macrophages, monocytes, myeloid and plasmacytoid dendritic cells (DCs), and myeloid derived suppressor cells (MDSC).
RNA Sequencing and TcR Repertoire Analysis
In collaboration with the SDRC’s Genomics Core, we offer sorting of immune cells in bulk or as single cells and determination of their gene expression profiles via RNA sequencing. This includes determination of the T cell receptor (TcR) repertoire usage of T cells from patients with or at-risk for diabetes, including T cells specific for b cell Ags. The HIMC also offers RNA and DNA extraction, Bioanalyzer services, gene expression and miRNA microarrays, and microfluidic qPCR arrays for human.
Beta Cell Antigens (Future)
Pending purchase of some requisite equipment, we will purify whole human proinsulin protein (and potentially other b cell Ags) for use in identification of b cell Ag-specific B and T cells from individuals with or at-risk for diabetes and determining their function. We will offer more information once these Ags are available.
Identification of Leukocyte Subsets in Human Islets
In collaboration with the Stanford Islet Research Core (SIRC), we can obtain human islets, make single cell suspensions and then determine the frequency and phenotype of immune cells extant in human islets via either flow cytometry or CyTOF.
Instruction and Consultation
DIMC staff would be happy to consult to determine the needs of individual investigators and to discuss the optimal assays for their studies.