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New Flow Cytometry Protocols

Here are some new protocols designed to cover basic areas of flow cytometry.  These are intended as general guides, not specific SOPs, which you can generate according to your own needs.

Cryopreservation of PBMC:  Download Protocol-Cryopreservation.pdf

Cell-surface staining for flow cytometry:  Download protocolsurface_staining.pdf

Activation for functional assays:  Download Protocol-Activation.pdf

Intracellular staining:  Download protocolic_staining.pdf

Experiment setup (for BD digital instruments):  Download protocolexp_setup.pdf

NEW Experiment setup (for BD analog instruments):  Download protocolexp_setupanalog.pdf

Posted on 30 July 2008 in Protocols and Notes | Permalink | Comments (1)

Detecting CD107 and IFNg

Many labs are measuring degranulation of T cells by cell-surface expression of CD107 (see Betts et al., J Immunol Methods 281: 65 (2003), Link to PubMed abstract), and it's often desirable to simultaneously detect intracellular cytokines, such as IFNg.  Unfortunately, optimization of both of these readouts in the same stimulation is not trivial.  I suggest the following tips:

1.  Stimulate cells in the presence of 5 ug/mL EACH of brefeldin A and monensin.  Monensin helps maximize the CD107 readout, whereas brefeldin A helps maximize the IFNg readout.  To acheive these final concentrations without addition of an excessive amount of solvent, dissolve monensin (Sigma #5273) at 5 mg/mL in methanol, then dilute 1:10 in PBS on the day of use, followed by 1:100 dilution into the culture medium.  Ditto with brefeldin A, using DMSO as the initial solvent (or use the FastImmune brefeldin A from BD [Link to product description], which comes as a 5 mg/mL stock in DMSO).

2.  Use antibody to CD107a (use of CD107b antibody adds only slightly to the signal) in PE [Link to product description] or PE-Cy5 [Link to product description], as these give the brightest signals.

3.  Add the antibody, at 10 uL per 200 uL of cell stimulation culture, at the beginning of the activation period, and activate for 5-6 hours.  Continue with processing for surface and intracellular staining as usual.

Posted on 31 May 2006 in Protocols and Notes | Permalink | Comments (21)

Hints for non-human primate CFC

We've published a techniques paper that addresses sample handling and other issues for macaque CFC (Link to PubMed abstract).  Here are answers to a few common questions:

How should samples best be handled, if they can't be stimulated on the day of harvest?  We suggest isolating PBMC on the day of harvest (BD CPT tubes work well for this), shipping the PBMC overnight, and stimulating the next day.  Stimulations can be done in a timed water bath, cooling to <18 C after the desired stimulation time of 6-10 hours, with processing done the next day.

What about plates versus tubes?  For human PBMC, we routinely use 96-well plates and the results are just as good as in tubes (see Download suniplate_cfcbmci.pdf).  We haven't yet done the comparison with non-human primate cells.

What are the best cytokines to monitor?  In general, in rhesus:  TNFa > IFNg > IL-2.  We haven't tried Th2 cytokines much.  The clones are important, especially for IFNg, where the B27 clone from BD Pharmingen is much superior to any others we've tested.

Posted on 29 March 2006 in Protocols and Notes | Permalink | Comments (0)

Peptide Mix Antigens

Overlapping peptide mixes are commercially available from vendors like Jerini Ag in Germany (http://www.jpt.com/index.htm) or BD Pharmingen (see Download peptidemix_hotlines.pdf).  These are commonly designed as 15 aa in length, with 11 aa overlaps, a paradigm pioneered by Florian Kern and colleagues (see for example Kern et al., 1999).  The utility of this approach, and comparison with mixes of 9 and 20 aa peptides, can be found in our JIM paper from 2001 (Link to PubMed abstract).  Here are some frequently asked questions:

How many peptides can be mixed together in a single stimulation?  The answer seems to be "many", maybe up to 300 or so, without significant competition for MHC sites.  However, as a practical matter, peptides are usually solubilized in DMSO, and there is a limit to their solubility (determined largely by sequence).  More than 0.5% final concentration of DMSO can cause toxicity to lymphocytes in a 6 hour assay.  So you may be limited on how many peptides you can mix together based simply on DMSO concentration.

What purity of peptides is required?  We use 80% purity as a standard, but this is more to ensure roughly equal representation of different peptides in a mix, rather than a requirement for performance.  We have not been able to see a difference in stimulation capability between purified and crude versions of a single epitope peptide for CMV.

Do you order them pre-mixed?  We have been able to get manufacturers to pre-mix and aliquot lyophilized mixes, and to QC these mixes for us.  This is much more convenient than solubilizing individual peptides and then mixing them, and allows for higher per-peptide concentrations when dissolving the final mix.  Vendors that offer pre-mixing, QC, and aliquoting include CPC Scientific (http://www.cpcscientific.com/) and American Peptide Company (http://www.americanpeptide.com/).

How do you stimulate with peptides?  Brefeldin A can be added at time 0 without detriment, because internal processing of peptides is not required.  6 hour stimulation is standard, but this can be lengthened up to 12 hours or more with increasing intensity of cytokine staining, before toxicity of the brefeldin A begins to interfere.

How do 15-mers stimulate CD8 responses?  We don't know for sure, but it seems likely that cell-surface and/or serum-derived proteases play a role in clipping longer peptides to generate species that can bind efficiently to class I MHC molecules.  Internalization does not seem to be required, as stimulation is not inhibited by chloroquine or inhibitors of TAP processing.

What concentration of peptide is required?  We typically use 1-2 ug/mL of each peptide in the mix.  The optimal concentration will actually be donor-dependent, since some donors will respond to high-affinity epitopes and/or epitopes that are easily presented, while other donors will not...so this is just a compromise recommendation.

Posted on 29 March 2006 in Protocols and Notes | Permalink | Comments (0)