cell bio lab

B cell activation steps

Ag bings to BCR, activates B cell, proliferates, differentiates into plasma B cell, plasma cells produce IgM Abs

Ist Ab to be secreted during infection

IgM

Most abundant Ab

IgG

Dilution formula

C1v1=c2v2

AA with ring structures

Tyrosine, phenylalanine, tryptophan

cell culture medium examples

DMEM, TCM, RPMI 1640

what does cell culture contain

inorganic salts, vitamins, sugar/glucose, buffered pH, phenol red indicator, AA, hormones and growth factors, fetal bovine serum, sodium pyruvate, non essential AA, L glutamine, HEPES buffer

how do we feed cell cultures so they can proliferate and grow

cell culture medium

hormones in cell culture medium

insulin- for glucose

transferrin- for iron binding

growth factors

optimal cell culture growth conditions

37 degrees Celsius, humid atmosphere, proper CO2 content to maintain buffering system (5-8%)

two types of cell cultures

primary explants and continuous cell lines

primary explants

cells derived from tissue or blood, the primary culture reaches a certain density then they have contact inhibition, and at that point you can transfer some to make a secondary culture

continuous cell lines

established cultures of cells essentially the same type, will not stop growing after contact inhibition, due to genetic modification using plasmid

outcome of cell growth- somatic cells vs transformed cell line

primary somatic cells enter senescence after 12 weeks roughly (due to telomere shortening after many cell cycles), primary cell culture can last 12 weeks, so when you transform the cells into continuous cell line they can last many years

types of cell lines

nontransformed- normal, have limited lifespans, we won’t use in the lab

transformed- genetically modified to last but still has normal functions of a cell, altered to show continuous growth

cancer cell lines- derived from cancers, shows continuous growth, may have alter functions due to being a cancer cell

cell characteristics- adherence and examples

non adherent- like blood leukocytes

adherent- like epithelial, endothelial, fibroblast, neuronal

epithelial cell characteristics

skin, intestinal/colon mucosa, liver or other organs, usually grow flat and spread out

fibroblasts

connective tissue, grow as a spindle shape

endothelial

line the blood vessels, grow spreading and flat

3T3 cells

mouse fibroblast transformed cells, don’t have contact inhibition

HT29

human colon carcinoma, cancer cells, they don’t have contact inhibition but may have modified function

IEC-6

rat small intestinal epithelial cells, non transformed

Caco-2

human colon carcinoma cells isolated from cancer

how do we avoid contamination

sterilize solutions and equipment, aseptic or sterile techniques, laminar flow hood, antimicrobials

3 methods for sterilizing solutions and equipment

autoclave- use high pressure and temperature to kill microbes

filtration- pass solutions through a membrane with a small pore size

irradiation- X rays or gamma rays to kill microbes (not UV bc it will change the nutrients in the culture medium)

aseptic or sterile technique examples

gloves, lab coat, ethanol spray

laminar flow hood purpose

circulates the air, turn on the hoods before using them, must work in the center of the hood

antimicrobials purpose in cell cultures

to reduce bacterial load, not for sterilization

penicillin- kills gram pos bacteria

streptomycin- kills gram neg bacteria

gentamicin- broad spectrum

amphotericin B- anti fungal

none are antivirals, so virus and mycoplasm bacteria with no cell wall can be a problem

role of trypsin/EDTA

EDTA secretes metal ions to enhance the cleaving ability of trypsin, which breaks the connections between cells and adherent surface

how to determine if cells are trypsinized

cells become round and bright instead of flat

role of trypan blue

stains dead cells blue

hemocytometer and how to use

a dyed cell counter, use trypan blue, pipet cells into hemocytometer, 10 microliters each side, count cells in each quadrant up to 100 cells, finish the number of cells in the last quadrant

dilution for hemocytometer

10 microliters cells, 80 of medium, 10 trypan blue

formula for percent viable cells

percent viable cells = viable cells/all cells

formula for viable cells/mL

viable cells/mL = (total viable/number of quadrants counted) x dilution factor x 10^4

why do we need viable cells/mL

to set up new cell cultures, to prepare dilutions

unregulated cell death

necrosis

necrosis

energy independent cell death, cell is damaged and then swells and bursts, releases intracellular contents, enzymes, DNA, and this release can damage nearby cells and activate the inflammatory response

regulated cell death types

apoptosis, autophagy, necroptosis, pyropoptosis, ferroptosis

ferroptosis

iron dependent cell death, driven by lipid peroxidation, not mediated by caspases, ROS accumulate in the cell

pyropoptosis

highly inflammatory mode of cell death, common in immune cells, is activated by infection from microbe/virus inside the cell, caspases are activated and pores are formed on the cell membrane, IL-1B and IL-18 cytokines are released causing the inflammatory response

necroptosis

programmed form of necrosis, is triggered if the apoptosis system is blocked, causes membrane rupture and inflammation

autophagy

cell eats itself, lysosome dependent degradation, provides nutrients during stress, maintains cellular homeostasis, is normally a helpful system but can lead to cell death if excessive, GLP-1s slow down digestion so much that cells do this

apoptosis

cell death without the release of cellular contents, is energy dependent, no nearby inflammatory response, important in development bc its used to build the nervous system and shape body parts, removes old cells in the epithelium, removes cells after immune response, kills infected cells

morphological changes: chromatin condenses, DNA begins to fragment into 200 bp pieces, nuclear envelop disassembles, cytoskeleton breaks, cell blebs forming bodies that can be phagocytosed, once caspase activation begins, cell will die no matter what

quantifying apoptotic cells

• run a gel to see how many fragments, does it form a ladder

• look for the activation of apoptosis specific proteases/caspases

• electron microscopy for cell blebbing

• stain the cells for condensed DNA

  • Syto24 stain- binds to DNA, can see if its in tiny blebs or in a blebbing cell

inducing apoptosis in cells

serum starve the cells causes in 2-3 days

why do we favor serial dilutions

there is less error than direct dilutions

serial dilution

making a smaller dilution ratio and then using that to create your final dilution

calculating dilution factor

the reciprocal of the fraction of the dilution

cell confluence

the cells are touching each other, no gaps between cells

importance of standard deviation

values are significant if values do not overlap with mean +/- SD

how to determine apoptosis

Syto 24 dye and epifluorescent microscope- stains nuclei in cells to view blebbing