Exam 3: Ch 9 Culturing Cells & Microscopy Flashcards Preview

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Flashcards in Exam 3: Ch 9 Culturing Cells & Microscopy Deck (63):
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culturing

maintaining isolated cells in the lab under conditions that permit their survival and growth

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3 advantages of cultured cells

grow cells of a single type

control experimental conditions

single cell easily grown into a colony of identical cells

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clone

strain of identical cells grown from a single cell

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immunoflurorescence microscopy

allows localization of specific proteins within fixed cells

provides a static image of their location

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chimeric protein

protein of interest covalently linked to a naturally fluorescent protein

allows visualization of movements of the protein in live cells

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organelle

membrane limited compartment of a cell

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culture medium

nutrient rich liquid

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cell-adhesion molecules

cell surface proteins that allow cells to bind to each other and to the ECM

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primary cell culture

a culture where the cell-cell and cell-surface interactions are broken by protease and chelation

fibroblasts become predominant cell (they lay ECM)

finite life span

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embryonic stem cells

can give rise to all tissues during development

can be cultured indefinitely

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oncogenic transformation

cells that are able to grow indefinitely (happens spontaneously)

a culture of immortal cells like this is a cell line

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flow cytometer

desired cell type labeled with an ab + attached fluorescent dye

cells flow past a laser beam that measures the emitted fluorescence to quantify # of cells of desired type in a mix

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fluorescence activated cell sorter (FACS)

a flow cytometer that can analyze the cells and sort them

ex. purify T cells from others (T cells have CD3 and THy1.2(

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apical

top surface

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basal

bottom surface that contacts underlying ECM called basal lamina

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Madin-Darby Canine Kidney (MDCK)

cells grown in special containers to study epithelial cellsq

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hybrid cells called ______ produce abundant monoclonal antibodies

hybridomas

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antibody

protein that binds to foreign molecules and tags them for elimination

made by memory B cells

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B cells are made where and by what

in the bone marrow from stem cells

genes undergo recombination during maturation to increase variety of Ab genes

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Ab are on the surface of _ cells

B

if antigen binds to the B cells, gets activated to make memory B cells

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memory B cells

make Ab if bound to antigen

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can a foreign molecule have multiple epitopes?

yes, reason for making polyclonal Ab

multiple B cells attach

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polyclonal

multiple clones of B cells

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monoclonal

one clone of B cells

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how to make a monoclonal Ab

inject mice with antigen, kill mouse at 2 wks, remove spleen, separate B cells w/ assay and choose Ab w/ best affinity

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problem with monoclonal Ab

B cells stop dividing/Ab stop dividing eventually

solution: hybridoma (cancer cell + B cell cross)

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myeloma cell

immortal lymphocytes that are joined with a normal B cell to make a hybridoma

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selection medium

medium that permits growth of only hybridomas

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what can monoclonal Ab be used for

affinity chromatography

immunofluorescence microscopy

immunoblotting

medicine

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obejctive lens

lens closest to the speciment

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projection lens

ocular or eyepiece

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resolution

ability to distinguish btw two closely positioned objects

limit for a light microscope is .2 micrometers

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two types of microscopy

visible light

fluorescence

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visible light microscopy is ___ contrast

low

increase by staining or optical tricks

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phase contrast microscopy

increase contrast using a phase ring that reduces bent light

direct light passes through an area of phase plate and transmits a small percentage of light

depends on thickness of specimen and difference in refractive index

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differential interference contrast microscopy (DIC)

increase contrast using a shadow effect with a condenser filter

method of choice for visualizing small details and thick objects

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dark field

no specimen = see blackness

specimen sparkling in light

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why stain?

live cells lack compounds that absorb light, and are invisible to a light microscope

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light and electron microscopy use stains that...

cross-link most proteins and nucleic acids

ex. formaldehyde

cut into sections 50 micrometers thick

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a molecule is said to be fluorescent if

it absorbs light at one wavelength and emits light at a specific and longer wavelength

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fluorescent microscope staining

get higher quality image (tag important part in thick specimen)

use UV light as excitation source for UV tags -- visualize emitted fluorescence

living or dead cells

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fluorochrome

fluorescent dye

ex. fura-2 sensitive to Ca2+, SNARF-1 sensitive to H+

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immunofluorescence microscopy can be used to detect specific _____ in fixed cells

proteins -- have Ab with fluorescent dye attached

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immunofluorescence microscopy

Ab covalently linked to fluorochrome like Texas red or green fluorescent protein

when bound to antigen, lights up

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reporter gene

glow tells us where gene is active

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lower E = ______ wavelength

longer

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goat anti-rabbit

indirect immunofluorescence

immunize a goat with Fc common to all rabbits to get better fluorescence upon binding

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epitope tag

cDNA encoding a recombinant protein fused to a short sequence of aa

when expressed in cells, the cDNA will generate the protein linked to the tag

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2 problems with fluorescent microscopy

light emitted from above and below plane of focus gives blurry image

to visualize thick specimens, serial images must be collected and reconstructed by computer (time-consuming)

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zebrafish microscopy

have clear embryo

fluorescence tagging used to see what grows where in growing embryo

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deconvolution microscopy

collect into overnight and computer takes out blurry info

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confocal microscopy

uses optical methods to obtain images from a specific focal plane and exclude light from others (otherwise image degraded)

light through pinhole

laser scanning

spinning disk

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focal plane

sliver of a thick specimen

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laser scanning confocal microscopy (LSCM)

eliminate out of focus info using a laser to limit light

multiple pics to make a stack of pics

Z series = multiple planes... 3D image

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total internal reflection fluorescence (TIRF)

visualize a thin focal plane like kinetics of microtubules and actin filaments

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fluorescence resonance energy transfer (FRET)

used to test if 2 molecules are next to each other

use 2 fluorochromes of different color where emitted light of 1st excites 2nd

illumination of 1st fluorochrome produces 2nd light if nxt to each other

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super resolution microscopy is a subset of ________ microscopy

fluorescence

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photo activated localization microscopy (PALM)

uses a type of GFP that only fluoresces when activated by a specific wavelength of light, different from its excitation wavelength

look at 1 molecule at a time

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strategy x

slightly different times of excitation and release

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electron microscopy

electromagnetic lens focuses a high-velocity electron beam

living material cannot be visualized

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transmission electron microscopy

visualize things that have been negatively stained with heavy metal

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cryoelectron microscopy

view biological specimens using TEM if the specimen is frozen

ex. virus capsids

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scanning electron microscopy (SEM)

view surfaces of unsectioned metal-coated specimens

limited by thickness of metal coat