Exam 3: Ch 9 Culturing Cells & Microscopy Flashcards
culturing
maintaining isolated cells in the lab under conditions that permit their survival and growth
3 advantages of cultured cells
grow cells of a single type
control experimental conditions
single cell easily grown into a colony of identical cells
clone
strain of identical cells grown from a single cell
immunoflurorescence microscopy
allows localization of specific proteins within fixed cells
provides a static image of their location
chimeric protein
protein of interest covalently linked to a naturally fluorescent protein
allows visualization of movements of the protein in live cells
organelle
membrane limited compartment of a cell
culture medium
nutrient rich liquid
cell-adhesion molecules
cell surface proteins that allow cells to bind to each other and to the ECM
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
embryonic stem cells
can give rise to all tissues during development
can be cultured indefinitely
oncogenic transformation
cells that are able to grow indefinitely (happens spontaneously)
a culture of immortal cells like this is a cell line
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
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(
apical
top surface
basal
bottom surface that contacts underlying ECM called basal lamina
Madin-Darby Canine Kidney (MDCK)
cells grown in special containers to study epithelial cellsq
hybrid cells called ______ produce abundant monoclonal antibodies
hybridomas
antibody
protein that binds to foreign molecules and tags them for elimination
made by memory B cells
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
Ab are on the surface of _ cells
B
if antigen binds to the B cells, gets activated to make memory B cells
memory B cells
make Ab if bound to antigen
can a foreign molecule have multiple epitopes?
yes, reason for making polyclonal Ab
multiple B cells attach
polyclonal
multiple clones of B cells
monoclonal
one clone of B cells
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
problem with monoclonal Ab
B cells stop dividing/Ab stop dividing eventually
solution: hybridoma (cancer cell + B cell cross)
myeloma cell
immortal lymphocytes that are joined with a normal B cell to make a hybridoma
selection medium
medium that permits growth of only hybridomas
what can monoclonal Ab be used for
affinity chromatography
immunofluorescence microscopy
immunoblotting
medicine
obejctive lens
lens closest to the speciment
projection lens
ocular or eyepiece
resolution
ability to distinguish btw two closely positioned objects
limit for a light microscope is .2 micrometers
two types of microscopy
visible light
fluorescence
visible light microscopy is ___ contrast
low
increase by staining or optical tricks
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
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
dark field
no specimen = see blackness
specimen sparkling in light
why stain?
live cells lack compounds that absorb light, and are invisible to a light microscope
light and electron microscopy use stains that…
cross-link most proteins and nucleic acids
ex. formaldehyde
cut into sections 50 micrometers thick
a molecule is said to be fluorescent if
it absorbs light at one wavelength and emits light at a specific and longer wavelength
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
fluorochrome
fluorescent dye
ex. fura-2 sensitive to Ca2+, SNARF-1 sensitive to H+
immunofluorescence microscopy can be used to detect specific _____ in fixed cells
proteins – have Ab with fluorescent dye attached
immunofluorescence microscopy
Ab covalently linked to fluorochrome like Texas red or green fluorescent protein
when bound to antigen, lights up
reporter gene
glow tells us where gene is active
lower E = ______ wavelength
longer
goat anti-rabbit
indirect immunofluorescence
immunize a goat with Fc common to all rabbits to get better fluorescence upon binding
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
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)
zebrafish microscopy
have clear embryo
fluorescence tagging used to see what grows where in growing embryo
deconvolution microscopy
collect into overnight and computer takes out blurry info
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
focal plane
sliver of a thick specimen
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
total internal reflection fluorescence (TIRF)
visualize a thin focal plane like kinetics of microtubules and actin filaments
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
super resolution microscopy is a subset of ________ microscopy
fluorescence
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
strategy x
slightly different times of excitation and release
electron microscopy
electromagnetic lens focuses a high-velocity electron beam
living material cannot be visualized
transmission electron microscopy
visualize things that have been negatively stained with heavy metal
cryoelectron microscopy
view biological specimens using TEM if the specimen is frozen
ex. virus capsids
scanning electron microscopy (SEM)
view surfaces of unsectioned metal-coated specimens
limited by thickness of metal coat
