Cells of interest to immunologists are often genetically heterogeneous (e.g., memory B cells, NK cells, cancer cells), and therefore single cell genetic analysis can provide exciting new insights. Flow cytometry provides a high throughput and extremely accurate front end for subsequent molecular analysis of single cells and can be considered as key method for immunologists. Applications include infectious disease and virology topics such as determining the percentage of infected cells within a cell population for diseases such as EBV and HIV, and studying the immune response of cells infected by pathogens such as Malaria and Dengue fever.

With its highly innovative Advalytix products Olympus provides a Simple, Seamless, and Sensitive® workflow for genetic analysis of FACS sorted single cells that has become very popular with immunologists. For mixing cells during the sort Olympus has developed the CellEvator a drop-in sample mixer for the BD FACSAria™ which uses proprietary surface acoustic wave (SAW) mixing technology to enhance sample mixing during cell sorting. This technology allows gentle, non-contact mixing that does not harm life cells.
For molecular analysis of single cells after high throughput cell sorting like single cell PCR, RT-PCR, qPCR, or sequencing Olympus offers the AmpliGrid system providing robust results with simple workflows. Our AmpliGrid slides provide a high placement accuracy of above 95%. Moreover, due to its two-dimensional layout, correct cell placement of single cells can easily be verified visually before moving on to molecular analysis (also available as automated solution). Despite its 1µL assay volume the AmpliGrid reaction delivers sufficient amplicon material to analyze your single cell molecular data via gel, capillary electrophoresis, sequencing, or qPCR. For clinical diagnostic applications, the AmpliGrid’s 1µL volume saves reagent costs for expensive detection kits. To minimize effects of sample manipulation on gene expression, Olympus developed AmpliCell, an innovative microculture slide chamber, which will be launched in October. With AmpliCell cells can be grown and directly analyzed by PCR without need of sample preparation.
Advalytix offers detailed protocols and technical support to help you port your current analysis method into the of 1µL world AmpliGrid. In addition, our
5 or 20-plex RT-PCR/ PCR multiplex primer design service is perfectly suited to help you elucidating immunology pathways.

Application examples:

• Dr. Rob Barrington (formerly with the Center for Biomedical Research at Harvard Medical School, now with Advalytix) is able to differentiate sub-groups of extremely rare mouse memory B cells using the AmpliGrid system.

• Dr. Collette Cywes (Channing Laboratories and Harvard Medical School) uses AmpliGrid to study single cells in a throat infection model.

• Norman Häfner and Matthias Dürst (Gynaekologische Molekularbiologie, Frauenklinik FSU Jena) reliably detect single HPV copies in single cells in a background of 1,000 non-infected cells