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inverted CommasI can’t recommend FloCyte enough to people. I wish everyone who prepares flow samples – even if they don’t run them – would take FloCyte’s courses

 

 

DNA Courses

Basics, Multiparameter Cell cycle, Cell cycle specific proteins, and apoptosis

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FloCyte has several possible programs for DNA studies, a Multiparameter DNA Cell Cycle module, with Cell specific Proteins / cytokines, and Apoptosis. These can be coupled with a software training program on Modfit from Verity.

 

Basics of Cell Cycle Analysis

 

  • A very brief history of cell cycle
  • Going from cell cycle to single parameter DNA histogram
  • What do DNA histograms tell us – what other information is required for interpretation?
  • Brief discussion of DNA dyes – do’s and don’ts
  • Examples of perturbations in DNA histograms
  • Sub-G1 cells – how to identify apoptotic cells from debris

 

Multiparameter Cell Cycle

 

  • BrdU to more precisely determine S phase
  • Traditional method
  • SBIP method
  • Other assays for proliferation – Ki-67, cyclins
  • Early two parameter approaches
  • DNA versus protein
  • DNA versus RNA
    • Native versus Denatured DNA for identification of mitosis


Use of cyclins to study cell cycle

 

  • Background on role of cyclins, cdks and ckis
  • Discussion of each of the useful cyclins: D, E, A, and B
  • Use of cyclins to study:
    • Refinement of cell cycle compartments
    • Drug treatment
    • Unbalanced growth
    • Drug point of action – stathmokinesis
    • Difference between cancer and normal cells
  • Three parameter approaches using cyclins and DNA
  • Multiparameter approaches using modification of chromatin proteins
  • Phosphorylation of histone H3 to identify mitotic cells
  • Phosphorylation of histone H2AX to identify DNA double-strand breaks
  • Sensitivity of the assay
  • Detection of DNA damage by a variety of clastogens
  • Measuring DNA damage using ATM
  • Other multiparameter approaches
  • Inactivated versus total retinoblastoma protein


Our apoptosis curriculum covers the basic understanding of what is happening in programmed cell death vs necrosis, and how to measure this using flow Cytometry as a tool.  Topics covered include

 

  • Tunel Assays
  • Caspases and how they relate to flow, and the assays which measure them, DEVD-Rhod110 and D2R for example
  • Annexin V
  • Mitochondrial DNA Probes such as Rhodamine123, CMXRos, JC-1, MitoTracker Green
  • Toxins
  • CFSE for cell growth/division and adding Hoechst for cell cycle in conjunction with CFSE to show halt in progression of cell cycle
  • Hoechst dual emission shifts – red shift is an apoptotic indicator, as indicated by nuclear “blebbing” and DNA laddering
  • And Multiplexing – using several assays such as Hoechst, annexin, CMXRos, for example to demonstrate apoptosis.