WEBINAR – Phenotypic Flow Cytometric Analysis of Regulated Cell Death
- 124 subpopulations show why Flow not Western blot
Currently, the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. Dr. Gary Warnes and his coworkers have shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3− ve), necroptosis (RIP3Hi + ve/Caspase-3− ve) and RIP1-dependent apoptosis (Caspase-3+ ve/RIP3+ ve) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations.
In this webinar, Dr. Gary Warnes will share with you and discuss this new approach to Regulated Cell Death (RCD) research that is recently published in Apoptosis journal and should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations. This study and related experimental aspects will be discussed in details, and the critical tips and tricks will be shared.
Key topics to be covered
- RIP3 and Caspase-3 intracellular labelling with a fixable live/dead probe allows the flow cytometric detection of necroptosis (RIP3high+ve/Caspase-3-ve) , apoptosis (RIP3-ve/Caspase-3+ve) and RIP1-dependent apoptosis (RIP3+ve/Caspase-3+ve)
- Further labeling with LC3B allows the detection of autophagic cells
Additional labeling with H2AX and PARP allows further identification of DNA damage, hyper-action of PARP and parthanatos
- Incidences of RCD were modulated by the use of zVAD and Necrostain-1
- Autophagy was shown to protect cells by reducing DNA damage
Wednesday, April 24, 2019
- 9 AM, San Francisco
- 12 PM, New York
- 4 PM, London
About the Presenter
Gary Warnes, Ph.D.
Flow Cytometry Core Facility Manager, Queen Mary University of London
Gary Warnes started working with flow cytometers in 1986 working on the AZT Concorde trial at St Mary’s and St Thomas’ hospitals. During Ph.D. studies flow cytometric use of Quantum Simply Cellular beads was used to calculate antigen numbers of CD69 and CD25 to highlight the immunosuppressive effects of Factor VIII blood products. While at St Thomas’ a flow cytometry assay was developed to show the degree of bacterial resistance to antibiotics (International Protocol, ‘94). In post-doctoral work in the US, the same flow cytometric assay was used to measure the immune response during sepsis for monocyte Tissue Factor, CD62P, CTLA-4, CD40, CD80, CD86 and CD154, published in the British Journal of Haematology, 1998. After working at the MRC and Cancer Research UK Imaging and Flow Cytometry Core Facilities then moving to the Blizard Institute in 2006, numerous assays have been developed and published including analysing oncosis, autophagy, reticulophagy, mitophagy, ER stress, necroptosis, classic apoptosis, RIP1-dependent apoptosis, DNA Damage, H2AX hyper-activation of PARP and parthanatos.