Molecular Cell Biology Lab Exam 2

cell culture

isolating cells from animal or plant and growing them in a favorable artificial environment

primary culture

stage of the culture after the cells are isolated from the tissue and proliferated under appropriate conditions

confluent

cells occupy the entire substrate surface and have no room left for expansion

subcultured

cells that have been transferred from one culture vessel to another to allow for further growth

-allows for further progagation of a cell line

-also known as passaging or reseeding

cell line

a population of cells that have been cultured and are maintained over time, often derived from a primary culture.

finite cells

normal cells that divide only a limited number of times before losing their ability to proliferate

continuous cell line

transformed cells that acquire the ability to divide indefinitely

transformation can occur spontaneously or be chemically or virally induced

fibroblast-like cells

bipolar or multipolar, have elongated shapes and grow attached to the substrate

epithelial-like cells

polygonal in shape with more regular dimensions and grow attached to a substrate in discrete patches

lymphoblast-like cells

spherical in shape and usually grown in suspension without attaching to a surface

adherent cells

monolayers on an artificial surface

suspension cells

free floating in culture medium

flasks

used to grow large batches, contain filtered caps to allow CO2 exchange

well plates

used for smaller batches, individual control over the wells

petri dishes

generally used with bacterial cultering, often solid medium is used

medium

designed to suppoert the growth of a population of cells; amino acids, inorganic salts, vitamins and sometimes antibiotics

serum

source of adhesion factors, nutritional, hormonal and growth factors; fetal bovine serum (FBS) is most common

buffering system

maintain the pH of the medium; sodium bicarbonate is most common

culture conditions; pH

varies according to the species or type of cell used, most normal mammalian cell lines grow well at pH 7.4

Culture Conditions; CO2

buffers against changes in pH through organic or CO2-bicarbonate

Culture Conditions; Temperature

depends on the body temperature of the host from which the cells were isolated, most mammalian cells grow best at 37 celsius

incubator

provides the appropriate environment for cell growth; CO2 - 5%, humidity - water in incubator, temperature - 37 celsius

laminar flow hoods

provide an aspetic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures

clean benches

only protect the cultures from the worker and are used if the cultures are harmless

biological safety cabinets (BSCs)

used to protect the cultures from the worker and the worker from the cultures

BSCs; Class 1

protects the worker and the environment but not the cultures

BSCs; Class II

protects the worker, the environment, and the cultures

BSCs: Class III

provides the highest level of protection to worker, environment and cultures through gas tight design

sterile work area;

disinfect surface before and after use; wipe the work surface with 70% ethanol, UV light can also be used to sterile the air and work surface

aspetic technique

gloves are sterilized with 70% ethanol, keep hands inside the hood while working. Change the pipette tip with each new material, never uncover a sterile vessel until you are ready to use it and cover immediately when done

glioblastoma cells

most common type of brain tumor

has poor prognosis and high mortality due to its highly invasive behavior, surgery is the major therapeutic method

DMEM

Dulbecco’s Modified Eagle’s Medium; contains vitamins, amino acids and glucose, 10% FBS, 1% penicillin-streptomycin

which cell type is polygonal with regular dimensions and grows attached to a substrate in discrete patches

epithelial cells

True/False: well plates are used to grow small batches of cells and provide individual control over each well

true

what type of sterile work environment protects the worker and environment, but not the cultures

class I

True/False: Good aseptic technique does not require individuals to spray gloved hands with 70% ethanol prior to entering the hood

false

confluence

% of a culture dish that is covered by adhered cells

-typically subculture at 80-90% confluency

medium exhaustion

indicated by color change due to pH drop

lactic acid builds up due to cellular metabolism (lowers pH, lactic acid can be toxic)

lag phase

cells become accustomed to their new environment

log phase

exponential cell growth (reproduction). production of new cells > death of cells

stationary phase

production of new cells is equal to death of cells

death phase

nutrients are exhausted; waste has built up. death of cells > production of new cells

techniques for dissociating adherent cells from the surface of the culture vessel

shake off, scraping, enzymatic dissociation (utilizes proteases)

protease

enzyme that digest proteins, dissociates cells from the vessel surface

trypsin-EDTA

is a proteast, extended activity can kill the cell, a chelator that binds to calcium and magnesium ions that may otherwise inhibit the trypsin activity

DMEM + FBS

inhibits trypsin, preventing cell death

Phosphate buffer saline (PBS)

used to wash cells in between steps to rid of unwanted media, isotonic so it will not lyse cells, cannot use water as it may kill cells

True/False: During the lag phase, the cell population experiences exponential growth

false

Why is FBS required when using trypsin

prevents cell death by inhibiting trypsin

three main components of cytoskeleton review

microtubules (made of tubulin), microfilaments (made of actin), intermediate filaments (made of keratin)

Actin

component of cytoskeleton that supports cell structure and allows movement of cells

found in two forms: F-actin (filamentous actin) and G-actin (globular actin)

Paraformaldehyde (PFA)

creates cross links between free amino groups in proteins

attaches cells to substrate without compromising morphology

excessively long incubations will affect cell integrity

Triton X-100

detergent solution

permeabilizes the membranes of cells

Rhodamine Phalloidin

probe with high affinity for F-actin conjugated to a red-orange fluorescent dye

Rhodamine

family of fluorone dyes, red-orange color

Phalloidin

bycyclic peptide belonging to a family of toxins isolated from the “death cap” mushrooms, binds to F-actin with high selecivity

DAPI

family of blue fluorescent dyes used to stain DNA, used to label the nucleus

how does paraformaldehyde fix cells

creates a cross line between free amino groups and proteins, attaches cells to substrate without compromising morphology

True/False, DAPI is used to label ribosomes

false

what form of actin does phalloidin have a high affinity for

F-actin

what color dyes are rhodamine and DAPI

rhodamine: red-orange, DAPI: blue

cell viability

the number of healthy cells in a sample

alamarBlue assay

a non toxic cell viability indicator for all cell types in both suspension and adherent cells

resazurin

active ingradient in alamar blue assay, blue low fluorescence when oxidized before entering cell

in healthy cells is it reduced to resorufin= produces a bright red/pink high fluorescence

reduction of resazurin takes place where

mitochondria

absorbance measurement in alamarblue assay

fluorescene is measured through spectrophotometry

healthy, live cells reduced at 570 nm, damaged/nonviable cells oxidized at 600 nm

the reduction of resazurin to resorufin is most affect by two variables

cell density: the greater the number of cells, the faster the reaction

incubation time: the longer the incubation time, the more complete the reaction

True/False, the alamarblue assay is used to measure viability in adherent cultures only

false

what type of instrumentation is used to measure the reduction of resazurin

spectrophotmetry

what color is resazurin before being reduced

blue

how is resazurin reduced

Reduced in healthy cells in the mitochondria

what is the reduced form of resazurin called

resorufin

immunocytochemistry

a technique for the visualization of proteins and peptides in cells using biomolecules capable of binding to the protein of interest

enzyme-coupled antibody

reporter example: antibody-peroxidase + DAB

visualization: brown color

specificity: antigen

fluorophore-labeled antibody

reporter example: antibody-Cy3

visualization: green excitation/yellow emission

specificity: antigen

biospecific small molecule dye

reporter example: DAPI

visualization: UV excitation/blue emission

specificity: DNA

immunocytochemistry steps

1. cell culturing

2. immunostaining

3. imaging

4. image analysis

direct method

one reporter labeled antibody is used, not sensitive enough for most proteins

indirect method

multiple binding of secondary antibodies to the same primary antibody, increased sensitivity, amplification of the signal

how antibodies are made

1. inject antigen into rabbit

2. antigen activates B cells

3. plasma B cells produce polyclonal antibodies

4. obtain antiserum from rabbit containing polyclonal antibodies

primary antibodies

produced in species that are different than the species of your sample, this prevents cross-reactivity of endogenous immunoglobulins in host species

secondary antibodies

developed to target the host species of the primary antibody you are using, under optimal conditions, this specifically binds to the primary antibody

primary antibody for the lab

glial fibrillary acidic protein (GFAP) antibody

-mouse monoclonal antibody

-recognizes and binds to human GFAP

secondary antibody for the lab

alexa fluor 488 affini pure goat anti-mouse

raised in goat, recognizes and binds to any antibody raised in mouse

-has a green fluorescent dye conjugated to the secondary antiobody

true/false, direct detection utlizes reporter labaled secondary antibodies

false

a scientist uses an antibody produced in rabbit to target a human protein. what should their secondary antibody target

secondary anitbody must target the host species of the primary antibody and be produced in an animal species other than the primary antibody’s host species