midterm (copy)

choosing the appropriate pipette

depends on volume youre transfering, the nature of the liquid (density, vapor, pressure, viscosity, surface tension) and the required degree of accuracy

Pasteur pipette

are made of glass and have a long tapered end that allows access to small soaces

serological pipettes

glass or plastic, available in a wide variety of sizes and are graduated which allow for transfer of specific volumes

transfer pipettes

made of plastic and contain volume markers which allow one to estimate volume aspirated and dispensed. only used if accuracy is not critical

piston driven air displacement pipettes

work horses of lab pipe tying typically used for dispensing accurate volumes less than 1 ml. has dial and specific tips.

volumetric pipettes

glass pipettes designed to accurately transfer a specified volume. liquid is aspirated with a suction device and dispensed by force of gravity. designed to deliver or to contain a specific volume

capillary pipettes

are much like volumetric pipettes but are intended for very small volumes the liquid is aspirated by capillary action and dispenses by the force of gravity may be graduated or intended for a set volume

we mainly use which pipettes

serological and piston driven

pipetting requires _____ and ____

precision and accuracy

accuracy

closeness of a measurement to a standard

Precision

closeness of individual measurements to eachother

range of pipette

the smaller the range the more accurate. find the smallest range for the volume you need

aspiration

pulling up

expulsion

pushing out

what's the key word to serological pipette

slowwww

serial dilution

substance in solution is diluted in a stepwise manner using a dilution factor that is constant at each step

why are serial dilutions prepared for experiments

things that require the use of concentration curves or trend lines and are also useful in procedures for plating cells

R^2 value can be used to determine how

the linear line fits the data- directly related to your accuracy

density of water

1 g/mL

in vivo

performed in a living organism- answer questions about multiple organs or organ systems

what makes a good model system

relatively inexpensive, easy to maintain, entire genome has been sequenced, have systems that are homologous to more complicated eukaryotic organisms including humans

ex vivo

scientific processes being performed on cells, tissues, or organs outside of the body

cell free systems

do not contain whole cells and have provided information about the roles of the proteins involved in membrane trafficking, proteins that bind to the membrane to initiate vesicle formation, and proteins responsible for cargo selection

in vitro model systems

cultured cells and cell free systems

cultured cells mimic

the activity of cells in living organisms

primary cells

cells that are harvested and used immediately for experimentation

cell line

genetically identical cells that divide indefinitely and can be cultured in the laboratory

HeLa cells

famous cancer cells from Henrietta Lacks

cultured cells have allowed scientists to

mimic the activity of cells in living organisms

Senesence

cells stop dividing

Cho cells

Chinese Hamsters Ovary Cells

Cho cells are ____ for many alleles

hemizygous

hemizygous

only one of two alleles is functional

basal media

provides vitamins, amino acids, fatty acids, lipids, carbs, trace elements, necessary for cell growth.

basal media we used

Dulbecco's Modified Eagle Medium and Hams F-12 media

serum

provides final concentration, vitamins, hormones, growth factors etc.

Serum we used

FBS (fetal bovine serum)

additives

penicillin/streptomycin

temperature

37 C

humidity

flask with semi-permiable membrane to allow for gas exchange- pan of water in bottom of incubator

carbon dioxide

5% and sodium bicarbonate buffer to help with acidity

Splitting

cells multiply, and have to be divided so they can continue multiplying. cho cells need to be split 2x per week

CHO cells morphology

grow in a monolayer, contact inhibited, polygonal shape, large centralized nucleus

light microscopy

visible light and lenses to create magnified lenses. requires stains. fixed cells.

Phase-contrast microscopy (PCM)

converts light phase shifts to changes in brightness int he image that is observed. no stains or fixation needed.

electron microscopy

most powerful type. uses a beam of electrons accelerated for illumination.

Niewmann-Pick-C1

responsible for the export of lysosomal cholesterol

Basophillic

bluish-purple

acidophillic

red

neutrophillic

pink or neutral

most common acidophillic stain

eosin

hematoxylin

a nucelic acid binding stain which imparts a pale purple color to the cytoplasm and a darker purple color to the nucleus

Oil Red O

stains lipids red

H&E

hematoxylin and eosin- a dye combination used to stain pathology specimens

Masson's trichrome stain

hematoxylin, acid fuchsin, aniline blue

silver nitrate

stains neurons black

Fluoresence Microscopes do what?

view "glowing" bacteria that are tagged with fluorescent dyes

Hoescht 33258

fluorescent dye

fragmented nucleus indicates a cell that is

undergoing apoptosis

staining typically ____ cells

kills

vital stains

chemicals that can stain living cells without killing them

confluence

% of surface covered in cells

epithelial cells

skin cells that cover the outside of the body and line the internal surfaces of organs- flattened plate like cells with a hexagonal shape.

methylene blue

a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color. stains DNA, RNA, and other negatively charged molecules

when working in the hood

wash gloves and all items with isopropyl, leave door lowered, dont cross hands or go in and out excessively, never open anything outside of cabinet.

contaminated media turns

yellow

Trypsin

makes cells dissociate form plastic side of container

Trypan Blue

a viability stain used to differentiate dead cells (blue) from living cells (clear)

PBS

phosphate buffered saline- like a wash rinse for the cells to remove the media so that trypsin can work

media contains the anecdote for

trypsin

cultured cells will not be ______ for observation

stained

epithelial cell

vessels for cell culture

flasks, culture dishes, multi well plates, chamber slides.

knowing surface area of vessel is important to determine

number of cells in the vessel

passaged

split

splitting cells is necessary to

maintaining the quality and phenotype of the culture

steps of cell splitting

disruption of cell monolayer, trituration of the cells, discarding a portion of the cell suspension resulting in the disposal of unwanted cells, and replating a small percentage of the homogenous cell suspension containing the desired number of cells

cell spreading/ cell seeding

making multiple vessels out of one original vessel of cells. important how many cells are transferred. that number is dependent on

- confluency required for the experiment
- doubling time of the particular cell line
- amount of time between seeding and performing the experiment.

size of our flasks

25 cm^2- polystyrene, Tissue culture treated surface

if the flask filter gets wet

the cells suffocate

Trituate

The process of mechanically mixing a material ( pipetting cells up and down to break up clumps)

UV light sterilizer

Utilizes UV light to kill bacteria in a dry setting

our cells must be split

2x per week

BSS

balanced salt solution ph of 7.4

trypsin

An enzyme that catalyses the extracellular breakdown of proteins. and stops cells ability to adhere to the surface or eachother

trypan blue

only appears in dead cells (cells with a compromised membrane)

cellometer

automatic cell counter

hemocytometer / advantages

Instrument used in counting blood cells- can distinguish between dead and alive cells

each 1mm^2 corner of a hemocytometer represents

total volume of 10^-4/cm^3

cells/ml =

average number of cells per square x dilution factor x conversion factor

pellets need to be frozen at

-80 degrees C

review hemocytometer cell count calculations

yay

H&E staining

hematoxylin and eosin

Hand E is most comonly used for

histology and cytology applications

H and E can be used to determine if a cell is

undergoing apoptosis or necrosis

apoptosis inducers

tunicamycin and UV light

cells are fixed with

formaldehyde

fixation

kills the cells, but preserves their morphology

mounting media

substance that is highly dense and has a high refractive index

mounting media we used

glycerol