Institute for Immunity, Transplantation and Infection

The Human Immune Monitoring Center (HIMC)

For more information

Contact:

Rosemary Fernandez, Flow Cytometry Manager         
Email » [rfernan]

Human Immune Monitoring Center
Fairchild Science Bldg, D033
299 Campus Drive
Stanford, CA 94305-5124

phone:  650-723-4984
fax:  650-498-7495

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:

  T cell Treg B cell DC/mono/NK Th1/2/17
FITC
live/dead
live/dead
live/dead
live/dead
live/dead
PE
CCR7
CD25
CD24
CD56
CXCR3
PerCP-Cy5.5
CD4
CD4
CD19
CD123
CD4
PE-Cy7
CD45RA
CCR4
CD27
CD11c
CCR6
APC
CD38
CD127
CD38
CD16
CD38
APC-H7
CD8
CD45RO
CD20
CD3+19+20
CD8
V450
CD3
CD3
CD3
CD14
CD3
V500
HLA-DR
HLA-DR
IgD
HLA-DR
HLA-DR

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.

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:

  cytokine panel BCR/TCR panel
stimuli
IFN-alpha
anti-IgM+IgG
IFN-gamma
CD3+CD28
IL-2
 
IL-6
 
IL-7
 
IL-10
 
IL-21
 
readouts
pSTAT-1
pErk1/2
pSTAT-3
pp38
pSTAT-5
pPLCgamma2
cell types

B cells, T cells, monocytes, non-T/B (NK).  Optional:  CD45RA (for naive vs memory T cells)

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:

  T cell ICS panel
stimuli
CD3+CD8
peptides or other stimuli of your choice
readouts
IFN-gamma
IL-2
IL-4 (or optionally, IL-22)
IL-17
TNF-alpha
cell types

CD3+CD4+ and CD3+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.

Protocols

1.  Short protocol descriptions, suitable for publications or grant applications:

Flow Cytometry Immunophenotyping:  PBMC were thawed in warm media, washed twice and resuspended at 1x10^7 viable cells/mL. 50 uL cells per well were stained for 45 min at room temperature with the antibodies shown in the table [above] (all reagents from BD Biosciences, San Jose, CA). Cells were washed three times with FACS buffer (PBS supplemented with 2% FBS and 0.1% sodium azide), and resuspended in 200 uL FACS buffer. 100,000 lymphocytes per sample were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v9.3 by gating on live cells based on forward versus side scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating.

Phosphoepitope Flow Cytometry (Cytokine stimulation, pSTAT readouts):  PBMC were thawed in warm media, washed twice and resuspended at 0.5x10^6 viable cells/mL. 200 uL of cells were plated per well in 96-well deep-well plates. After resting for 1 hour at 37C, cells were stimulated by adding 50 ul of cytokine (IFNa, IFNg, IL-6, IL-7, IL-10, IL-2, or IL-21) and incubated at 37°C for 15 minutes. The PBMCs were then fixed with paraformaldeyde, permeableized with methanol, and kept at -80C overnight. Each well was bar-coded using a combination of Pacific Orange and Alexa-750 dyes (Invitrogen, Carlsbad, CA) and pooled in tubes. The cells were washed with FACS buffer (PBS supplemented with 2% FBS and 0.1% soium azide), and stained with the following antibodies (all from BD Biosciences, San Jose, CA): CD3 Pacific Blue, CD4 PerCP-Cy5.5, CD20 PerCp-Cy5.5, CD33 PE-Cy7, CD45RA Qdot 605, pSTAT-1 AlexaFluor488, pSTAT-3 AlexaFluor647, pSTAT-5 PE. The samples were then washed and resuspended in FACS buffer. 100,000 cells per stimulation condition were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v9.3 by gating on live cells based on forward versus side scatter profiles, then on singlets using forward scatter area versus height, followed by cell subset-specific gating.

Intracellular cytokine staining:  PBMC were thawed in warm media, washed twice and resuspended at 1x10^7 viable cells/mL. 200 uL of cells were plated per well in 96-well V-bottom plates. After overnight rest at 37C, activation reagents [specify here] + 10 ug/mL brefeldin A (Sigma, St. Louis, MO) were added and the cells incubated for 6 hours at 37°C. EDTA was then added to 15 mM final concentration, and the cells washed with PBS. They were then stained with Red LIVE/DEAD cell viability dye (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After washing with FACS buffer (PBS supplemented with 2% FBS and 0.1% sodium azide), cells were resuspended in 200 uL of 1x BD FACS Lysing Solution (BD Biosciences), and the cells incubated for 10 min at room temperature. They were then centrifuged at 500 x G for 5 min, and resuspended in 200 uL of 1x BD FACS Permeabilizing Solution 2 (BD Biosciences). After 10 min at room temperature, the cells were washed twice with FACS buffer, and resuspended in 200 uL FACS buffer. Staining with cytokine-specific antibodies [insert list here] was then performed for 1 h at room temperature, followed by two additional washes with FACS buffer. 100,000 CD3+ cells per sample were collected using DIVA 6.0 software on an LSRII flow cytometer (BD Biosciences). Data analysis was performed using FlowJo v9.3 by gating on live cells based on forward versus side scatter profiles, then on singlets using forward scatter area versus height, followed by dead cell exclusion using Red LIVE/DEAD viability dye, and then cell subset-specific gating.

Activation reagents: PMA, PHA, Ionomycin (Sigma, St. Louis, MO); anti-CD3/CD28 antibodies (Invitrogen, Carlsbad, CA); CMV pp65 + IE-1 peptide mix (JPT, Berlin, Germany).

Standard Ab cocktail: CD3 V500, CD8 V450, CD4 PerCP Cy5.5, IFNgamma FITC, IL17 PE, IL2 PE Cy7, TNF AlexaFluor 700, IL-22 APC.

2.  Detailed protocols and addtional information:

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