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Molecular Cell Biology > Lab test 2 > Flashcards

Lab test 2 Flashcards

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1
Q

How can cells be obtained?

A

(enzymatic or mechanical means before cultivation

a) grinding tissue to disaggregate it
b) disaggregation by enzyme

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2
Q

Adherent cells

A

a) anchorage-dependent
b) propagate as a monolayer attached to cell cx vessel
ex) fibroblast, astrocytes

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3
Q

Suspension cells

A

a) can survive and proliferate without being attached to a substratum
ex) hematopoietic cells, some malignant tumor cells

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4
Q

Finite cell line

A
  • cells usually divide only a limited number of times before losing their ability to proliferate
  • cell lines whose telomeres get shortened each replication
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5
Q

Continuous cell line

A

-cell lines are immortal which can occur spontaneously or can be chemically or virally induced

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6
Q

What are applications of cell culture?

A
  1. modeling systems for studying normal physiology and biochemistry of cells
  2. drug screening & development
  3. large scale manufacturing of biological compounds such as vaccines, therapeutic proteins
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7
Q

Cell Cx hood

A
  • laminar flow hood
  • biosafety cabinet
  • used to reduce the presence of airborne contaminates
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8
Q

Incubator (humid CO2 incubator recommended)

A
  • required to maintain optimal carbon dioxide concentration and temperature
  • 5% Co2 (the concentration found in the blood)
  • 37 degrees Celsius
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9
Q

telomers

A
  • function is to protect the actual important DNA
  • found on the end of DNA sequences
  • long span of repeating sequence that gets cut every replication
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10
Q

What are basic requirements of cell culture

A
  1. cell cx hood, incubator, water bath, centrifuge, refrigerator & freezer, microscope, sterilizer, vessels, pipettes, media
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11
Q

Mammalian cell base media

A

DMEM

  • most commonly used medium for mammalian cells
  • contains about 4x as much vitamins and AAs present in the original formula & two to 4 times as much glucose
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12
Q

Mammalian cell complete media components

A
  • DMEM
  • ABx
  • FBS serum
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13
Q

FBS

A
  • Fetal bovine serum
  • contains large number of growth promoting activities
  • it buffers toxic nutrients by binding them
  • it neutralizes trypsin and other proteases
  • it contains peptide hormones/hormone like growth factors that promote healthy growth
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14
Q

Typical cell culture media mix

A
  1. salts
  2. amino acids
  3. sugar
  4. vitamins
  5. serum
  6. antibiotics
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15
Q

Aseptic hood technique

A
  1. gloves
  2. 70% ethanol spray
  3. completly in hood
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16
Q

PBS

A

phosphate buffer solution

  • salt solution
  • use to wash cells
  • has same osmalarity of mammalian cells
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17
Q

Cell seeding

A

transfer of a predetermined concentration of cells to a new vessel

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18
Q

Confluence

A

refers to the proportion of the surface which is covered by cells

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19
Q

Cell passaging

A
  • process of transferring cells to new vessels providing more media and space
  • done when they reach to 80-90% confluency in flask/dishes/plates
  • cells are typically passaged before becoming fully confluent in order to maintain their proliferation
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20
Q

Steps of cell passaging

A
  1. remove old media from original plate using serological pipette or new tip
  2. use a new tip to wash with PBS, then remove
  3. add 80 um of trypsin/edta from top of each well to cover surface of well
  4. incubate plate for 5 min with trypsin
  5. remove plate from incubator, add growth media to inhibit trypsin
  6. add whole solution of each well to a new and sterile centrifuge tube
  7. centrifuge solution
  8. remove surfactant
  9. break pellet and mix into solution
  10. perform cell count using hemocytometer
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21
Q

trypsin

A

protease used to remove cells from the surface of the plate

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22
Q

Rhodamine phalloidin stain components

A
  • has 2 components
    a) rhodamine
    b) phalloidin
  • a high affinity F-actin probe conjugated to TRITC
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23
Q

TRITC

A

tetramethylrhodamine

-red-orange fluorescent dye

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24
Q

Actin

A

-a component of the cytoskeletal system that allows movement of cells and cellular processes

25
Q

Rhodamine component of Rhodamine phalloidin stain

A
  • a family of related chemical compounds, fluorine dyes
  • fluorescent
  • inexpensive
  • TRITC is a rhodamine derivative used in Rhodamine phalloidin
26
Q

Phallodin component of rhodamine phalloidin stain

A
  • a bicyclic peptide belonging to a family of toxins isolated from the deadly Amanita phalloides (death cap)
  • Phallodin molecule binds to actin fibers
  • Fluorescently labeled phalloidin has virtually identical binding properties with actin from different species including plants and animals
27
Q

Rhodamine Phalloidin

A
  • selectively stains F-actin
  • superior to antibody staining
  • optimal for fixed and permeabilized samples
28
Q

How does rhodamine phalloidin dye work?

A
  • phalloidin binds f-actin with high selectivity while TRITC provides red-orange fluorescence
  • very specific staining
29
Q

Hoechst Fluorescent stain

A
  • blue fluorescent dye used to stain DNA
  • Bis-benzimides (2 rings)
  • excited by UV lights
  • compatible with antibodies and other probes labeled with rhodamine dyes
30
Q

What are the most commonly used Hoechst dyes?

A

33258 and Hoechst 33342; have similar excitation/emission spectra

31
Q

How does Hoechst stain dye?

A
  • binds preferentially to adenine thymine (AT) regions of DNA
  • requires 2 wavelengths of light for visualization: excitation as 540 nm and emission as 565 nm
32
Q

Paraformaldehyde

A

fixes cells

33
Q

Triton

A

permeabilizes cells so that hoechst stain can reach DNA

34
Q

Immunohistochemistry

A
  • using chemical molecules to visualize antigens by use of antibodies
  • makes it possible to visualize the distribution & localization of specific cellular components within a cell or tissue
35
Q

Antigen

A

any molecule that is foreign to a host, will induce immune response by the host
-can be part of something or may be the whole thing

36
Q

Antibody

A
  • a protein that binds specifically to antigens and induces immune response
  • foreign particles are marked as AB binds
  • glycoprotein that are produced by plasma cells
37
Q

How can antibodies be visualized

A

-tagged with stains or enzymes so that they can show as colored or induce a reaction that creates a stain

38
Q

Antibody binding

A

2 types: polyclonal & monoclonal

39
Q

Polyclonal antibodies

A

-large complex antigens may have multiple epitopes & elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies

40
Q

Monoclonal antibodies

A

antibodies specific for a single epitope and produced by a single clone are called monoclonal antibodies & are commonly raised in mice

41
Q

two types of antibody staining

A
  1. direct

2. indirect

42
Q

Direct antibody staining

A

-only have a primary antibody which is labeled

43
Q

Indirect

A

Have a primary antibody and a secondary antibody that is labeled. 2nd AB is attached to the tail of the primary antibody

44
Q

Ki67Ab

A
  • a primary antibody stain
  • uses Ki67 antigen
  • a nuclear protein
  • useful in establishing cell growth fraction in neoplasms
  • red color
45
Q

What is Ki67Ab specific to?

A
  • cells that are dividing only
  • must be in G1, S, G2, or M phases only
  • absent in resting G0 cells
46
Q

DAPI stain

A
  • similar to Hoechst stain
  • stains nucleus
  • not dependent on dividing or nondividing
  • blue
47
Q

fluorochromes

A

-a common label for antibodies

48
Q

applications of antibody labeling

A
  • cancer diagnostics
  • differential diagnostics
  • treatment of cancer
  • research
49
Q

General immunochemistry protocol

A
  1. fixation: using PFA
  2. permeabilize using triton X
  3. blocking of nonspecific sites by FBS
  4. primary antibody staining (Ki 67 Ab)
  5. Secondary Antibody staining (fluoroscence labelled)
50
Q

Alamar blue

A
  • a viability assay
  • uses natural reducing power of living cells to convert resazurin to the fluorescent molecule Resorufin
  • amount of fluorescence produced is proportional to the number of living cells
51
Q

viability assay

A
  • analyzes ability of organs/cells tissues to maintain or recover viability
  • basically determines the growth and proliferation of the cell
  • helps to quantify the number of live & dead cells
52
Q

Inactive ingrediant of alamarBlue and characteristics

A

resazurin: non toxic, cell permeable, blue, non fluorescnet

53
Q

What happens to resazurin as it enters the cell

A

resazurin is reduced to resorufin which produces a bright red fluorescence

54
Q

Advantages of Alamar Blue

A
  1. Superior performance
  2. fast and simple assay
  3. economical and safe to use
55
Q

Superior performance of alamar blue

A
  1. quantitative: provides accurate measurement over time
  2. high sensitivity an linearity: detects as few as 50 cells
  3. Robust performance: highly referenced for cytotoxicity and viability assays
56
Q

Why is alamar blue fast and simple?

A
  1. no cell lysis: ideal for use with time course experiments or post measurement functional assays
  2. flexibility: can be used with primary cells or cell lines with adherent cells and cell suspensions
  3. scalable: mix-and read, homogeneous assay enhances epeed while minimizing effort
    compatibile with fluorescence or absorbance based on instrumentation
57
Q

How is alamar blue safe and economical?

A
  1. Economical: requires less reagent to get results comparable to the competition
  2. Safe: nontoxic, nonradioactive reagent, safe for cells, user, and environment.
58
Q

Alamar blue process

A
  1. add alamar blue to cells
  2. incubate 1-4 hours
  3. read fluorescence or absorbance
  4. amount of fluorescence/absorbance is proportional to the number of living cells and corresponds to the cells metabolic activity
59
Q

What kind of alamar blue response do damaged & nonviable cells have ?

A

-lower signals response than healthy cells because cells have lower innate metabolic activity

Molecular Cell Biology (16 decks)

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