The detection of events as rare as 0.02% of total T cells requires both the design of suitably controlled experiments and a well-maintained flow cytometer. By following the protocols supplied with our products carefully, you should achieve successful staining. Below are suggestions to help you troubleshoot, should unsatisfactory staining be obtained. Please also consult the protocol optimization pages, or Pro5® MHC Pentamer handbook.
For expert guidance, the technical support team at ProImmune are all Ph.D-qualified Immunologists, with years of lab experience between them.
In order to achieve successful Pentamer staining, the following points should be noted:
Care should be taken with preparation of cells to maximize recovery and obtain a sufficient quantity of viable cells for staining. An excess of dead or dying cells will result in poor quality staining and increased background.
Flow cytometer instrument settings should be determined empirically for each different cytometer using samples of unstained cells and single-color controls. Forward scatter (FSC) and side scatter (SSC) voltages should be set using unstained cells so that the live lymphocyte population lies on scale (normally positioned at co-ordinates 400 and 200 respectively). Set a gate upon the live lymphocyte population, then set the voltages for each individual fluorescent channel so that the live cells are contained within the first log decade of each fluorescent channel.
Use single-color stained controls to adjust compensation settings for all the fluorescent channels required for the experiment. Due to overlapping emission spectra, some fluorescence from one fluorochrome may be detected in another channel. If the overlap is not corrected by compensation, the data will include the signal from each of the fluorochromes that has been ‘seen’ by the inappropriate detector. Set the compensation so that the center of the positively stained cell population lies in line with the center of the negative cell population and parallel with the appropriate axis.
Non-specific staining is defined as events seen in the CD8-negative, Pentamer-positive quadrant of antigen-specific cells, or in the CD8-positive, Pentamer-positive quadrant of negative control cells.
Background (non-specific) staining is sometimes observed when performing flow cytometric analysis of human and murine samples, particularly in the CD8-negative, Pentamer-positive quadrant. This problem is largely caused by non-specific staining of B cells, and can be reduced or eliminated by co-staining with anti-CD19 antibody and gating on the CD19-negative cells when performing analysis. Alternatively, anti-CD19 magnetic beads could be used to remove B cells prior to staining.
Application note: Elimination of non-specific staining (PDF)
The figure below demonstrates the effect of gating out B cells from murine splenocytes. 1 x 106 naive C57BL/6 (a)-(c) or SIINFEKL-immunized C57BL/6 (d)-(f) splenocytes were incubated with 1 test unlabeled H-2Kb/OVA-specific Pro5® MHC Pentamer (SIINFEKL) for 10 minutes at room temperature (22°C). Cells were washed, then further incubated with 1 test Pro5® R-PE Fluorotag, 1 test anti-mouse CD8-FITC (clone KT15) and 1 test anti-mouse CD19-PECy5 (clone 6D5) for 30 minutes at 4°C. In (a) & (d), the live splenocyte population shows considerable non-specific staining in the CD8-negative, Pentamer-positive quadrant; (b) & (e) A region R2 is set upon the CD19-negative cells; (c) & (f) The live splenocyte population is re-gated to show only events within R2 (which excludes B cells), and the non-specific staining has now been eliminated. This illustrates that a substantial majority of the non-specific staining can be attributed to B cells.
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Please note that mouse immunisation protocols need to be optimized to produce sufficient immune responses. Several rounds of immunization, with appropriate adjuvants, may be required to achieve a response that exceeds the threshold of detection.
Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis. Davis, MM et al Nature Immunology 2011 11:551 [PubMedID: 21760610]
Tracking epitope-specific T cells. Moon, JJ et al Nature Protocols 2009 4:565 [PubMedID: 19373228]
Tricks with tetramers: how to get the most from multimeric peptide-MHC. Wooldridge, L et al Immunology 2009 126:147 [PubMedID: 19125886]
Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative. Mallone, R et al Diabetes/Metabolism Research and Reviews 2011 27:720 [PubMedID: 22069251]
Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium. Britten, CM et alCancer Immunology and Immunotherapy 2009 58:1701 [PubMedID: 19259668]