L4: Apoptosis I part 2 Flashcards
flow cytometry?
looks at dna content inside the cell.
Sub-G1 Peak: In normal cells, the sub-G1 peak is low because apoptosis isn’t happening at a significant level.
Most normal cells will be in G1, S, or G2/M phases (with a normal distribution of DNA content).
Sub-G1 (fragmented DNA) is not prominent in normal cells.
Sub-G1 Peak: For apoptotic cells, the sub-G1 peak is higher than in normal cells. This is due to DNA fragmentation, where cells lose DNA during apoptosis (breaking into small fragments).
This sub-G1 peak represents the fraction of cells that have fragmented DNA (typically less than 2N DNA content), and thus it’s higher in apoptotic cells compared to normal cells.
this method uses Propidium Iodide – Fluorescent DNA stain that binds to DNA. The graph typically shows fluorescence intensity (x-axis) vs. cell count (y-axis). less intense fluorescence i think
tunnel assay?
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labelling
caspase activated dna can nick dna by cutting 1 strand a time. leaves free 3’ ends. terminal deoxynucleotidyl transferase (TdT) adds exogenous biotynilated nucleotides (UTP) to the free 3’ ends and incorporates into DNA. e.g: enzymes coupled to Streptavidin: Protein from Streptomyces (bacteria). Has high affinity for Biotin. they bind to labelled bases and enzymes generate a coloured reaction product.
apoptosis and disease?
cancer=cells escape apoptosis. neurodegeneration in parkinsons and ad. autoimmunity. failiure to make ag receptors. failure to suppress autoreactive immune cells. immune clearance: immune cells kill targets by inducing apoptosis, impaired killing of infected cells.
apoptosis in t and b lymphocyte development?
90-95% of T cell precursors die in thymus
90-95% of developing B cells die in bone marrow
Fail to produce antigen-specific receptors OR
Produce self-reactive receptors (activation-induced cell death)
If apoptosis is defective:
Autoimmune diseases (autoreactive T and B cells)
e.g. multiple sclerosis, type 1 diabetes
apoptosis in the immune system response?
cytotixic t cells bind to foreign antigens on class 1 mhc on infected cells. mhc binding to the tcr activates tc which secretes perforin and makes holes in the membrane and granzymes and granulysin enter that promote apoptosis.
