Lab 5: Flow Cytometry

Question 0

Early stages of apoptosis

Answer 0

Cell shrinkage, plasma membrane (blebbing), PS translocation.

Question 1

Apoptosis

Answer 1

Orderly, energy-dependent, highly regulated process for cell death. Differed from necrosis which is defined as degenerative, disorderly, random form of cell death.

Question 2

Apoptosis late stages

Answer 2

Mitochondria become leaky, organelles contract to form apoptotic bodies, chromatin condensation, DNA fragmentation.

Question 3

Apoptosis

Answer 3

Energy dependent- organized. Plasma membrane integrity maintained, ordered DNA degradation, immuno-suppressive (LPC). Cell elimination (PS).

Question 4

Necrosis

Answer 4

Bioenergetic impairment- disordered. Plasma membrane integrity lost, random DNA degradation, immuno-stimulating (HMGB1). Initiation of cell growth and tissue repair (HDGF).

Question 5

Assays for apoptosis

Answer 5

Cyt C release, PS relocation, membrane permeability, TUNEL (terminal transferase dUTP nick end labeling), DNA ladder formation,

Question 6

Annexin V-PE

Answer 6

Calcium -dependent PS binding protein. Bound by phycoerythrime which helps it distinguish between early and late apoptosis. (578 emition)

Question 7

Apoptosis late event

Answer 7

Membrane permeabilized, detected by 7-AAD which is a nonpermeate dye that stains cells with leaky and damaged membranes. Does not enter in tact cells or active cells. Identifies necrotic/ late apoptotic cells. Detection in the far red range allows for minimal spectral overlap with phycoerythrine.(650)

Question 8

Hydrodynamic focusing

Answer 8

Particles suspended in a highly pressurized stream fluid that travels through a narrowing chamber with velocity of the fluid greatest at the core channel and zero at the wall. Particles pass in a single file line.

Question 9

Side scatter

Answer 9

Cell structure and granular content. Reflection and refraction,

Question 10

Forward scatter

Answer 10

Cell size. Diffraction

Question 11

Fluorescent probe

Answer 11

Antibody conjugated to fluorochrome. Possible to analyze several parameters at once. Applications: cell surface receptors and intracellular organelle markers, immuniphenotyping, cell cycle and apoptosis studies.

Question 12

Limitations of FACS-based apoptosis

Answer 12

No unequivocal method to distinguish apoptotic from necrotic cells by FACS.
Final state of apoptosis (apoptotic bodies) is generally read as cell debri and can look like necrosis. Better to analyze cells at early apoptosis.

Question 13

Pharmacological applications of Guava Nexin Assay

Answer 13

Many anti-cancer drugs induce apoptosis. Guava Nexin can be used to screen large numbers of compounds for apoptotic activity, Guava Nexin can be used to quantitate the potency of an apoptosis-inducing drug (EC50).

Question 14

Negative controls

Answer 14

Media plus cells, and media with cells and 0.5% DMSO.

Question 15

Gating

Answer 15

Procedure to selectively visualize cells of interest while eliminating cell debris or dead cells, each dot is one cell. Move gates such that cell debris is excluded from other quadrants.

Question 16

Difference between EC50 and ED50

Answer 16

EC 50 refers to the concentration of drug that induces a response halfway between the baseline and maximum after a specific exposure time.

ED 50 is the dose of drug that produces a therapeutic response or desired effect in 50% of the subjects taking it.