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Flashcards in In vitro Analyses Deck (20)
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1

What is a primary cell?

it's isolation directly from tissue
morphologically and metabolically similar to parent tissue
can only divide a limited number of times (senescence)

2

What are immortalized/continuous cells?

mutated cell lines
derived from tumors
morphology and metabolism can differ from parent tissue

3

What are the seven components of a medium?

Liquid solution is the media
Provides nutrients to cells
Buffer
Environment (temp and concentration of CO2)
Phenol Red (pH indicator)
Antibiotics and Antimycotics
Serum

4

What is necrosis?

Damage to the cell is severe
In most cases, a rapid injury will result in necrosis
Characterized by ER swelling, membrane blobbing, membrane breakdown, organelle leakage, and random DNA degradation
Often affects groups of cells
Coupled to an inflammatory response
Causes: infection, toxins, trauma

5

What is apoptosis?

programmed cell death
the cell knows it's damaged so it will prepare for death

6

What are the characteristics of apoptosis?

chromatin condensation
cell shrinkage
preservation of organelles by cell membranes
engulfment by neighboring cells
membrane blebbing
small to no immune response

7

What are the two apoptotic pathways?

Intrinsic: the cell senses death in itself
Extrinsic: the cell is signaled from other cells to die

8

What is the caspase?

enzymes playing essential roles in programmed cell death and inflammation.

9

What is caspase staining?

detects the activity of the enzyme to determine whether or not a cell is undergoing death

10

What is Anexin V?

used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.
Determined with treating of staurosporine

11

What is Neutral Red?

a cell viability assay
vital dye (vital = able to penetrate living cell)
will only stain living cells
stains lysosomes
taken up by active transport

12

What is Calcein-AM?

a cell viability assay
fluorescent dye
taken up by active transport
partially metabolized by the cell
Calcein-AM in solution out of cell does not glow, converter to calcein and turns green

13

What is Propidium Iodide?

a cell death assay
intercalating agent (winds itself in DNA)
not membrane permeable (can only enter a broken membrane, hence death)
dye exclusion assay

14

What is Trypan Blue Exclusion Test?

a cell death assay
vital stain to selectively color dead tissues or cells blue. Live cells or tissues with intact cell membranes are not colored
can not pass through the cell membrane
can bind to serum proteins (not good)

15

What is CR51 Release?

tests the immune system to kill off cells
used heavily in tumor and viral cytosine studies
Target cells incubated with radio active Cr-51 then exposed to immune cells
If the immune cell kills the target, Cr-51 is released and then measured

16

What is the MTT assay?

a cell metabolism assay
NADPH reduces MTT into formazan (purple)

17

How is oxygen consumption measured?

fluorescent chemical is employed that is water soluble and membrane impermeable
quenched by O2
non destructive reaction
can be measured in real time
Flouresence decreased as O2 concentration increases

18

What is MEM Elution?

an in vivo assay
Minimum essental medium elution
employs L-929 cells (mouse fibroblasts)
incubate in the presence of material
looking for morphological changes and cell lysis
doesn't tell us about the death mechanism
If there is a cytotoxic effect, their is a drop in confluency

19

What are agar test used for?

Looks for zone of death or inhibition
Neutral red is used and followed by a wash
A cell monolayer of fibroblasts and cells of interest sit on dish, covered in agar with a test sample/pellet on top

20

What is genotoxicity?

standard is to use three different cell lines, at least two of which are mammalian
Bacterial mutagenicity - cheap, grow easily, smaller genome
looking at chromosomal structure and health
DNA destruction