exam 1 - cell culture

alternative names for biosafety cabinets

tissue culture hoods

laminar flow hoods

chemo hoods (pharmacy)

categorization of BSCs

class I, class II, class III (some have subtypes A, B, C)

laminar air flow

continuous

uniform in velocity and direction

flow pattern is parallel to solid surface

horizontal laminar flow hood

does not provide protection to personalle or environment; minimal risk

BSL 1

microbes are not known to cause disease

BSL 2

microbes are moderately pathogenic and can cause disease

BSL 3

microbes can be transmitted through air and inhaled

can cause serious and sometimes life-threatening diseases

BSL 4

microbes are highly contagious and extremely life-threatening

no treatments

BSC classifications

Class I - Class 4

Class I BSC Classification

BSL 1, 2, 3

personnel and environmental protection only

low to moderate risk biological agents

Class II BSC Classification

BSL 1, 2, 3

subclassifications A1, A2, B1, B2

sample, personnel, and environmental protection

low to moderate risk biological agents

Class III BSC Classification

BSL 4

sample, personnel, and environmental protection

high risk biological agents

acronym for HEPA filters

high efficiency particulate air filters

HEPA filters

can remove >99.9% of airborne particles =,>,< than 0.3 microns

pleated mats of randomly arranged fibers

affected by fiber diameter, thickness, and face velocity

catches air particles

how do HEPA filters catch air particles?

by interception, impaction, and diffusion

interception

particles w/ less mass stay w. air stream and impinge upon fiber as they go around it

impaction

particle inertia causes it to leave flow stream lines and impact on fiber

diffusion

Brownian motion/diffusion due to molecular bombardment

two types of CO2 incubators

water-jacketed

direct heat

inverted microscope

light source above the stage

objectives below stage

phase-contrast

what kind of water should you fill a water bath with?

distilled water

CHO

Chinese Hamster Ovary

immortalized cell line

anchorage-dependent (adherent)

RPMI

Roswell Park Memorial Institute

EDTA

ethylene diamine tetraacetic acid (e.g Ca, Mg, Fe, Cu)

EGTA

ethylene glycol tetraacetic acid

binds Ca w/ higher affinity than Mg

HT1080 cells

fibrous connective tissue

cancer cell line

anchorage-dependent (adherent)

attributes of cells

fibroblastic

epithelial

attributes of cells: fibroblastic

pointed, elongated, bipolar

attached cells

grow in sheets, loose contact

attributes of cells: epithelial

polygonal, more regular dimensions

attached cells

grow in sheets, close contact

what is the wide variety of complex media supplemented with fetal bovine serum (FBS)?

salts

carbohydrates

amino acids

vitamins

hormones

growth factors

water

what are usually added to medium + FBS?

antibiotics

buffers

sodium bicarbonate and HEPES

phenol red

pH indicator

yellow (acidic) → orange (neutral) → violet red (basic)

what does feeding cells do?

replenishes nutrients, eliminates waste/metabolites, maintains proper pH by replacing spent medium w/ fresh medium

DMEM

Dulbecco’s Modified Eagle’s Medium

components of DMEM

inorganic salts

amino acids

vitamins

other

add L-Glutamine

reasons to passage or subculture cells

increase cell numbers

seeding cultureware w/ adequate # of cells for experiments

preservation of cells or cryopreservation

cloning

characterization of cells

establishing a cell strain, cell selection

how do cells attach in vivo?

via extracellular matrix by specific cell surface receptors

calcium and glycoproteins on cell surface

how do cells attach in vitro?

adhere to plastic (polystyrene)

animal cell membranes have net (-) charge

treated cell cultureware has net (-) charge → excellent for attachment

how does ECM fibronectin and cells attach?

via integrins

how to release attached cells

• use divalent cation chelators like EDTA and EGTA

• use of protease - trypsin

• use chelator and protease together - trypsin-EDTA

ways to inactivate trypsin

adding serum

adding trypsin inhibitor

adding Ca and Mg

diluting the enzyme

placing cell suspension on ice

how to prepare detached cells for counting

inactivate trypsin

centrifuge at 200xG to pellet cells

resuspend cells in nutrient medium

ways to count cells

hemocytometer and automated/electronic cell counters

hemocytometer

use vital dyes or stains like Trypan Blue dye exclusion

• dead cells are stained blue

ways to get a cell count

hemocytometer

electronic counters

DNA assays

protein assays

glucose utilization

O2 utilization

expression of a product

what is needed for cell counts w/ a hemocytometer?

hemocytometer, hand tally, trypan blue

steps of cell counting

place cover slip

pipet 10 ul of cells suspended in trypan blue

equation for % viability

(# of viable cells/total # of cells) *100

equation for calculating number of cells/mL

# of cells/mL = total # of cells in 10 squares * dilution factor * 1,000

equation for calculating total # of cells in cell suspension

total # of cells = # of cells/mL you want to seed* total number of mL in cell