Cell Lab

what are the 2 uses for growing cells in a lab

clinical and practical biotechnology

what are primary cells taken from

derectly from tisue

what is the difference between primary cell lines and continuous cell lines

primary cell lines have a finite lifespan, continuous divide indefinetly

why would someone use/not use primary cell lines

because they closely resemble in vivo physiology, but they have the disadvantage of being a bit trickier to grow and ultimately die

why would someone use/not use continuous cell lines

they are easy to grow and divide indefinitely, however they are less representative of in vivo systems and will acquire increased levels of genetic modifications over time

what are the 2 ways both continuous and primary cell lines grow

adherent or in suspension

when will adherent continuous cells stop growing

when they reach the point where they form a solid monolayer with minimal space between the cells, even then other cell types might continue to grow on top of each other

what is 100% confluency

when adherent cells form a solid monolayer with minimal space between cells

what are HeLa, HEK293, SF9 examples of

common immortal cell lines used in labs

what is HeLa

the first human immortal cell line, derived from a cervical cancer tumor

what is HEK 293

human embryonic kidney epithelial cells

what is SF9

immortal insect cells that can be grown in suspension

how are risk groups of microorganisms, proteins and nucleic acids organized

by increasing danger from 1→4

who determines risk group classification for biological hazards

the public health agency of canada and the canadian food inspection agency

which risk group is capable of causing serious disease in humans but is unlikely to do so

RG2

which risk group is likely to cause serious disease in humans or animals for which there are effective treatments

RG3

which risk group is highly contagious and causes serious/fatal diseases for which there are no treatments

RG4

what is a containment level

minimum physical containment and operational practices required for safe handling of infectuous materials and toxins

what containment level are regular teachin labs

CL1

where are all RG2 pathogens contained

in CL2 facilities

what is the defining addition to a CL2 room

a biological safety cabinet (BSC) equipped with HEPA filters

what rooms at Dal are CL2

tissue culture rooms

what are added to rooms to make them CL3

primary and secondary barriers such as sealed windows and the use of a bsc for all work and strictly controlled access

how many CL3 labs does canada have

over 100

how many CL4 labs are in canada

2, both in winnepeg

what is the PPE needed for cell bio labs

lab coat

proper footwear

gloves

what is proper procedure for wearing gloves in cell bio labs

if you have a cut wear a bandaid under the glove

take them off so they are inside out when off

make sure they are not ripped

wash hands with soap and water once they have been taken off

what risk group are CHO cells

RG1

what is the difference between a bsc and a fume hood

a fume hood simply sucks air from the hood out a vent, a BSC creates an air curtain across the front opening which prevents aerosols from escaping the front and unfiltered air from opening, when air leaves it is exhausted through a high efficiency particulate air (HEPA) filter.

what is a HEPA filter

High efficiency particulate air filter, filters air coming in and out of a biological safety cabinet to keep the inside air and outside air seperate

what does a CO2 incubator do

short term storage and growing of cells, provides a humidified environment with a constant temperature and supply of CO2 (5%) which maintains the pH of cells at a physiological level

why are inverted microscopes necessary in cell biology

they have the lens on the bottom and the light source on top, this is necessary since cells usually grow on the bottom of the flask and there is often condensation at the top, additionally, the large stage provides a great base to view the large flasks

what does fetal bovine serum do and what is it normally found in

media typically contains 5-10% fetal bovine serum, this provides growth factors, hormones, nutrients and electrolytes that eukaryotic cells need to survive

what do antibiotics do in media

minimize risk of bacterial growth following contamination

why is media pink

due to the presence of the pH indicator phenol red

what happens if media becomes acidic/basic

acidic and it will turn orange

basic and it will turn purple

thanks to phenol red

what might a change in colour of the media pH indicator indicate

excess of metabolic by-products

cantaminated with bacteria, yeast or fungi

what is the procedure if there is a change in media colour due to excess of metabolic by-products

time to split the cells (passaging/subculturing) or change media to replenish depleted nutrients

what would a lab technician use to transfer a line of adherent cells from one vessel to another

trypsin to cleave the proteins in the cellular attachment points

what does trypsin do

cleaves cellular attatchment proteins so proteins float freely in a vessel

what is mixed into the media with cells for long term storage

DMSO

what does DMSO do

a special cryopreservation agent that reduces the formation of ice crystals

how are cells frozen for long term storage

using a special container filled with isopropyl alchohol which will cool cells at a rate of 1 degree/min, frozen in media with DMSO, when cells are fully frozen they are transfered to a liquid nitrogen Dewar for long term storage below -130 degrees

what was done to the cells before lab 1

cells were transfered into flasks so that they were adhered to the bottom by the time the lab started and were at the desired confluency

what does PBS do

washes the cells while keeping them at a constant pH

how would you seperate adherent cells from the media

dump media, wash in PBS, uncleave with trypsin, add fresh media, spin in microcentrifuge, dump supernatant

what is the cryoprotectant we use in the lab to test it’s effect on heat-shock proteins

DMSO

how do we measure live vs dead cell concentration

using a hemocytometer and trypan blue to dye the dead ones, then counting the 4 squares

what stain is used to determine cell viability

trypan blue

how does trypan blue work

dead/dying cells will allow the blue dye to leak into the intracellular space

what chemical is used in an exclusion test

trypan blue to determine cell viability

what colour will alive cells show in an exclusion test

grey/clear

what is the standard way to count cells

with a hemocytometer, cells are pipetted into a chamber on the top grid which makes them easy to count

which squares of a hemocytometer are counted

the corner 4

what is the equation for cell concentration

cell per ml=(number of cells counted/number of large squares containing counted cells (usually 4))x(dilution factor)x10^4

what is the equation used to calculate the dilution factor

total volume in tube/volume of sample

what setting (brightfield or phase contrast) is used to view specimens that have been stained or have their own natural colour

brightfield

what setting (phase contrast or brightfield) is used to view highly transparent specimens

phase contrast

how does phase contrast microscopy work

convert differences in light phase shifts into differences in light intensity which makes very thin, living, transparent specimens visible

what needs to align in a microscope to achieve phase contrast

phase plate and special filter in the turret

what position should the turret be set to for brightfield

O

what position should the turret be set to for phase contrast

Ph1

what is the variable tested in the multi week lab

temperature stress

what is the sample set to test the effect of the variable called

treatment

what is the sample that is not exposed to the variable called

negative control

what is the purpose of the negative control

serves as a baseline and will demonstrate what will happen in the absence of a variable

what were the 3 treatments that CHO cells underwent before lab 2

Cold shock=90 mins at 4 degrees

Heat shock=90 mins at 44 degrees

negative control=90 mins at 37 degrees (optimal)

what are the 2 kidns of techniques for protein extraction from cells

physical and chemical

what are the 3 methods of physical protein extraction from cells

liquid homogenization

sonification

manual grinding

what is liquid homogenization

a physical method for protein extraction, shears cells by forcing them through a narrow space

what is sonification

physical method for protein extraction, machine uses pulsed, high frequency sound waves to agitate and lyse cells, sound waves are delivered using a vibrating probe which is immersed into the cell suspension

what is manual grinding

physical method of protein extraction, works well to isolate proteins from plant cells, tissue is frozen then smashed which releases the proteins (i.e what i do to ur mom)

what is the challenge with physical methods of protein extraction

it is difficult to have consistent preparations from one day to the next, these methods can also generate heat which can cuse proteins to denature

how do chemical methods extract proteins from cells

using detergents of hypotonic solutions (low salt) to disrupt the phospholipid membrane and extract proteins

what is the detergent in RIPA buffer

triton x-100

what are the components of RIPA buffer

detergent called triton x-100

proteins and phosphatase inhibitors to ensure extracted proteins do not denature

what does RIPA buffer do

chemical method of extracting/seperating the proteins in cells from the cells themselves

what would you add to cells in order to seperate the proteins

RIPA buffer

if you spin RIPA buffer + cells, what will the pellet be at the end

the pellet will be all the non-protein parts of the cell while the proteins will be in the supernatant

how did we determine protein concentration in lab

bradford assays

how does a bradford assay work

bradford reagent binds to protein which causes a change in colour from brown to blue and the intensity of this colour can be measured using a spectrophotometer

how is the intensity of colour measured in a bradford assay

spectrophotometer

what is the generic protein standard to which unknown samples can be compared during a bradford assay

bovine serum albumin

what is the dilution series of a bradford assay

an abvious colour change made with bovine serum albumin which demonstrates the relationship between protein concentration and recorded absorption

how is the concentration of an unknown sample determined from it’S absorption and the standard curve of BSA

the equation of the line of the standard curve can be used to determine the protein concentration in an unknown sample, replace y with absorbance, find x

how do we figure out how much protein we were able to extract from the cell culture by RIPA buffer

mix bradford reagent with newly prepared protein lysate and get an absorbance reading then use the equation of the line of the standard curve of BSA

how is absorbance measured in a spectrophotometer

a photocel converts received light energy into electrical energy so the reading is displayed in units

what does the blank cuvette for a spectrophotometer contain

only pipes buffer and bradford reagent

what is in the cuvettes when they were measured by the spectrophotometer

protein lysate (proteins + RIPA buffer), pipes buffer, bradford reagent

what is the first step in determining protein expression

separate CHO proteins using gel electrophoresis and transfer them on a membrane

what is the common method for separating charged molecules

gel eletrophoresis

what does the gel in gel electrophoresis do

acts as a molecular sieve, alowing differential migration of proteins based on size

what are the gels that proteins are electrophoresed through made of

polyacrylamide

what is the Pa in SDS-PaGe

polyacrylamide

how are proteins separated in gel electrophoresis (by what measurement)

weight

what gel are DNA generally eletrophorosed through

agarose

how are DNA seperated in gel electrophoresis (by what measurement)

according to their number of base pairs

what molecules will find themselves closer to the bottom at the end of gel electrophoresis

smaller/lighter ones

what is mixed with cell lysate prior to gel electrophoresis

sample buffer

what is the negatively charged detergent in sample buffer

sodium dodecyl sulfate (SDS)