The Human Immune Monitoring Center (HIMC)

Flow Cytometry

Flow cytometry is used to assess immune cell phenotypes and functions.  We offer standardized immunophenotyping, phosphoepitope, and intracellular cytokine assays for human blood cells.  Our assays have all been optimized for use with cryopreserved PBMC; use with fresh samples must be coordinated in advance.

Immunophenotyping:  We have developed a standard set of surface marker panels for immunophenotyping of B, T, NK, DC, and monocyte subsets in cryopreserved PBMC.  These have been created as pre-formatted, lyophilized cocktails in 96-well plates, a technology which improves reagent stability and thus assay reproducibility, while also reducing labor time and the possibility of reagent addition errors.  These are the markers in our standard immunophenotyping panel:

If desired, up to two additional antibodies can be added to each of the five cocktails, subject to availability and compatibility.  We used the PE-Texas Red and AlexaFluor 700 dyes for expanded panels. Please contact Kavita Mathi at: kavitam@stanford.edu
for more information on Lyoplates and immunophenotyping.

Phopshoepitope Analysis:  We use antibodies speific for phosphorylated protein epitopes to interrogate signaling cascades, allowing for their analysis by flow cytometry. Defects in signaling may be associated with specific disease states, making this an ideal method for interrogating cellular responses.  We offer the following two standardized "phospho-flow" panels:

Intracellular cytokine staining (ICS):  Another short-term consequence of immune cell activation is the production of cytokines by responsive cells. The frequency of responsive cells and the types of cytokines they produce can be useful biomarkers for monitoring immune responses to vaccines or specific antigens, or to monitor immune function as related to disease progression. We have developed a standard 8-color flow cytometry panel for enumerating multiple cytokines on CD4+ and CD8+ T cells:

User Instructions

1.  Planning your assay:  All assays are run in batches of up to 12 samples.  If you have more than 12 samples, please try to randomize your batches so that, for example, cases and controls are not segregated into separate batches.

2.  Collecting your samples:  Please use an optimized CRYOPRESERVATION PROTOCOL for viable PBMC.  We recommend to freeze 10^7 cells per cryovial, and supply one cryovial for each of the above assays.  PBMC processing and cryopreservation services are also available through our Blood Processing Unit.

3.  Running your samples:  An online order is required before samples can be run. Turnaround time varies depending on the size of your project and the current workload; smaller projects can usually be completed within 3-4 weeks of order placement and sample delivery.

4.  Data analysis:  A standard analysis using FlowJo software is provided for all assays.  We will generate an Excel report of the analyzed data, as well as pdf's of the gating results, via our Basecamp project management system.  New users will recieve an email invitation to join this system.  Excel data is simultaneously uploaded to our online database, Stanford Data Miner, where it can be integrated with other HIMC data from your lab as well as clinical/demographic data, if provided.  Click the help link in the upper left corner of the Data Miner homepage for instructions on using this system.

Statistical consultation is available through the HIMC Statistical Consulting Service.

Protocols

Please see our Protocols page for complete lab protocols, as well as short descriptions suitable for publications and grant applications.