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Do you want to learn how to use flow cytometry in your research?

UCI is hosting a hands-on flow cytometry class with FloCyte that comprises 1-2 hours of lectures, summarizing how flow cytometry works and basic applications.  We will then spend the remaining time with a hands-on section where you can apply this knowledge to immunophenotyping blood samples.

Apr. 26, 2023

9 AM to 5 PM

Hosted by

UC Irvine

Bill & Sue Gross Stem Cell Center

Irvine, California

Course tuition $400

Contact Dana Buckman for Information

Dana@FloCyte.com

www.FloCyte.com

SDSC meeting Nov. 10, 2016

We have not had a local flow cytometry meeting since before Cyto. You can get 1 CEU for this meeting which is donated by FloCyte. ThermoFisher sponsored tonight’s meeting with talks focused on their flow cytometer, the Attune and RNA Prime. There were 2 sponsor talks of the evening and 1 talk from a local scientist at UCSD.
The first talk was from Nori Ueno about the newest innovations in eBioscience’s RNA Prime technology. eBioscience became a part of ThermoFisher this year when they were bought through the Affymetrix acquisition. RNA flow allows single cell analysis of both protein and RNA simultaneously. For some proteins in a cell, there aren’t very good antibodies. Using probes for the RNA for these proteins gives the ability to analyze them using flow cytometry. Previous RNA Prime assays allowed the researcher to look at 3 different RNA sequences in a cell, whereas the new version adds an extra RNA probe giving you access to 4 RNA targets and many other proteins that can be seen at the same time.
Cliff Ramsdell then gave a talk on the technology of the Attune and how it can overcome challenges in rare cell analysis. By focusing the cell stream using acoustics, it keeps the stream in the center of the laser thereby allowing an increase in speed without a decrease in CV due to the stream moving around past the laser. This reduces the manipulations that scientists need to do to analyze millions of cells in a short period of time.
ThermoFisher had a contest for scientists to win a new Attune based on a research challenge. Charles Prussak, director of the Cell Therapy Translational Laboratory at UCSD, won this award. His work in cancer research focuses on reversing checkpoint inhibitors and retrain the immune system to kill cancer cells. He is looking at the modulation of PD1, PD-L1, and CTLA4. He has found that by using a more physiological media to expand tumor infiltrating leukocytes (TILs), he can increase the cell number without changing the cell types extracted from the tumor. Their goal is to expand these cells and put them back into the patients using chimeric antigen receptor (CAR) T cell technology. By engineering the T cell to respond to the tumor cells, they can get around the checkpoint background in the tumor cells. They want to remove patient TILs, engineer in a single chain variable region to target them to the tumor cells, and keep them in culture for a maximum of 4 days causing the least amount of skewing of the populations before putting them back into the patient. He is using the Attune to study rare cells within the tumors for a better understanding of what they need to do to bring better therapy to cancer patients.
This was a fun evening of socializing with our peers in flow and great talks on how flow cytometry is helping in cancer research. Looking forward to a new series in 2017.

This was our first meeting of the year and we had a great turnout.  Everyone was very excited to hear about the new developments on the CyTOF instrument from Fluidigm.  There are 3 CyTOFs here in San Diego now.  Cheryl Kim introduced Tad George from Fluidigm who brought us up to date on the latest developments on the CyTOF and the software used to analyze the high complexity data.  They start with a program called Max Par which helps in designing the panels for flow depending on your target of interest and analyze using various programs such as SPADE and viSNE to help reduce the dimensionality to make the data understandable.  The big data generated from these large antibody panels to help elucidate cell how cells are behaving in many different types of tissues is daunting and these programs are allowing scientists to get a better grasp on the biology.

Raul Catena from the University of Zurich gave a fascinating talk about how they are developing a 3D reconstruction model based on the imaging mass cytometer.  Using modified equipment, they do serial sectioning of tumors, label them with antibodies to 44 different targets, and reconstruct the tumor data within their microenvironment.  One of the issues with doing tumor research on whole tumors using normal flow cytometry is that the tumors have to be fresh so they don’t have a large population of dead cells and the tumors need to be digested to get a single cell suspension.  Using the new technology of the imaging mass cytometer, even FFPE samples can be used, and the tumor does not need to be digested.  This allows the researcher to understand the microenvironment of the tumor and how cell populations change with different drug treatments.  They have a very low detection limit of 100 copies and a 1um resolution.  They are currently studying phosphorylation within the tumors to better understand what drug treatments will work in different tumors.  They are also developing better software to visualize and interpret the high complexity data.  Also in development is a way to understand where labeled drugs are going within the tumor.  Because of the use of metal tags in mass cytometry, they can label the drugs with specific metals and monitor where they go.

We look forward to the next SDSC meeting in a few months and following the exciting research using flow cytometry.

 

Tonight’s meeting began with Carina Torres introducing a few members to allow everyone to get to know each other.  Announcements included job postings for Miltenyi as well as CEUs being offered by FloCyte for these meetings.  FloCyte will also be offering more regular flow cytometry classes and wanted to get a better idea of what type of classes the community was interested in.  A survey will be sent out soon.

The first speaker for the day was from one of the sponsors, Miltenyi Biotec.  Matthew Drew talked about how Miltenyi has changed over the years, starting in Stefan Miltenyi’s mother’s basement.  They have grown from a sample prep company to offering over 600 flow cytometry antibodies, flow cytometers, and sorters.  They offer complete work flows for scientists to provide all of their reagent needs.

The next talk was from the 2^nd sponsor, DeNovo Software.  Kaya Ghosh is a scientist based in San Diego helping scientists learn how to use FCS Express software.  She went over the new capabilities version 5 offers.

The main speaker was Chris Groves from MedImmune, an AstraZeneca company.  They are creating a company-wide flow cytometry platform to allow their scientists to plan and analyze their flow cytometry experiments more accurately.  They provide what they call a global core which is a unified website that gives everyone access to protocols and assay planning features, among many others.  They have been working with Miltenyi to develop standard flow cytometry panels giving scientists access to easier ways to run their research.  They have created video tutorials, automated immunophenotyping methods, and multiassay toolkits.  Their goal is to help move research forward faster by giving everyone the same tools to run their assays.  If everyone uses the same platforms and assays, it is easier to standardize their work.  He showed some research done on a CAR-T agonist for cancer treatment.  They used a CT26 tumor model to study cells infiltrating the tumors.  Monitoring exhaustion markers, such as TIM3 and PD1 helped them monitor their drug treatments.  They plan to develop more tools for their scientists to do cutting edge research, such as using the MACSPlex miRNA beads from Miltenyi to study changes in tumors in response to treatments.  They are moving towards standardizing assays for their scientists to give them better tools to do better research.

Cheryl Kim ended the talk by announcing that the next SDSC meeting will be Dec. 3, 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.