SDSC June 2015

This quarter’s flow cytometry users’ group meeting took place at our normal site at the Eli Lilly conference center above Green Acre. Carina Torres, the flow cytometry core manager at Eli Lilly began the meeting by introducing new members to the group. This is such a close group and it is nice to welcome new members so we can get to know one another. She then explained that CEUs would be offered at future meetings through FloCyte by new owner, Dana Buckman. Scientists who need CEUs can get 2 credits for each meeting for both ICCE and CLS. A sign up column will appear on the GoogleDoc for future meetings so certificates can be prepared ahead of time. Cyto2015 is coming up at the end of June and very few of the members were going since it is in Glasgow this year. Next year, it will be in Seattle again making it more affordable for many scientists to go. ICCE is being offered right before the Cyto conference. The ICCE accreditation is becoming more important for a flow cytometrist’s career. It shows the scientific community of their advanced knowledge in this field. It is offered at other times around the country. Classes for this test are offered through FloCyte and ExCyte.

Matthew Cato from eBiosciences began his talk reviewing where they were a year and a half ago in respects to the RNA flow assay. He reviewed the benefits of looking at single cells to understand the biology versus looking at an average value within many cells. A protein of interest may be highly expressed on a very small subset of cells. When looking at a single marker on the cells, it may be hard to distinguish that small subset. Adding additional phenotyping markers help identify which population expresses the protein of interest making it easier to see. Using antibodies to proteins on a cell has helped in this field. eBiosciences has advanced their RNA flow to a point where they can look at RNA transcript very specifically. Using antibodies to the protein alongside monitoring the RNA transcript, they can watch the kinetics of when the protein is made and when the RNA is degraded. This is a very powerful technique. There are still no antibodies available for some proteins of interest. A way to be able to view these proteins of interest is to look at their RNA transcripts. This technique has opened up a lot of research interest for proteins, such as IL-21 or IL-23. This technique also eliminates the need for cytokine secretion blockers, such as monensin and brefeldin A. The new protocol of the PrimeFlow assay removes methanol, which had caused some antibody and fluorochrome issues previously. Their buffers are compatible with cytokines, transcription factors, and even phospho protein staining. This advanced assay has opened up a lot of exciting possibilities in regards to studying rare cell types, such as ILC2 cells involved in different cancers. Digesting tumors to find a rare infiltrating leukocyte has now become possible through the use of PrimeFlow.

Next up was Brent Kern from Pfizer. He recently submitted a paper to Cytometry on receptor occupancy and blocking of the IL-7R as a therapeutic for autoimmune disease. IL-7 circulates in the blood at very low concentrations (2pg/ml) and is essential for normal T and B cell development by activating the Jak/Stat pathway.   The IL7R is expressed on T cells and early B cells. Upon binding of IL-7, the receptor is shed and downregulated. The IL-7R has been found to be involved in multiple sclerosis (MS), type 1 diabetes, lupus, ulcerative colitis, and primary biliary cirrhosis. Blocking IL-7 binding should reduce the autoimmune response. Testing in EAE mice, a mouse model for MS, shows a decrease in MS type symptoms. By adding a competitive antibody to the IL-7R they showed a decrease in receptor occupancy as well as pSTAT signaling. They monitored internalization of the receptor using flow cytometry. After adding their blocking antibody, they showed that they could stop internalization of the receptor with a non-competing antibody to the receptor. They ran studies on cynomolgus monkeys and were able to show efficacy of their blocking antibody at doses as low as 0.3mg/kg. This data looks very promising and they are hoping to start human trials in the near future.

This was a very informative meeting and fun for the local flow cytometry community. It is a great opportunity for flow cytometrists to get to know one another and to learn how flow cytometry is helping in new areas of research.

See you at the next SDSC meeting in the fall.

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