Techniques for isolating primary culture isolation

Question 1

Difference between primary cell culture and cell lines?

Answer 1

Primary cell culture-are cells directly from the tissues and has finite lifespan, similar morphology and expression markers to the parent cell, is primary culture till it’s subcultured.
While cell lines have an infinite number of doubling populations, it is different from the parent cell,have to continuosly divide for at least 6 months.

Question 2

Cell lines have to be malignant

Answer 2

false

Question 3

A cell line established from a patient with a tumour has to be a cancer cell line

Answer 3

false

Question 4

What limits the cells growth when they are cultured?

Answer 4

cell cell contact, nutrient depletion, contact inhibition, accumulation of apoptotic cells

Question 5

What do you use to take iut adherent cells when splitting a passage?

Answer 5

trypsin

Question 6

What does dilution factor depend on primarily?

Answer 6

what type of cell line

Question 7

Why shouldn’t cells be cultured continuously?

Answer 7

Becasue of genetic/phenotypic drift and to avoid contamination

Question 8

What are the culture condition(o2,co2,temp)

Answer 8

o2-21%
co2-5%
37 degree
High humidity(reduce hypertonic)

Question 9

What is in the growth media?

Answer 9

Foetal calf serum(contains growth factor and hormones)

Standard salt mix, bicarbonate with CO2

Question 10

Defined cell lines use foetal calf serum

Answer 10

false, use EGF

Question 11

What must you do consistently when culturing cells?

Answer 11

Replenish and replace media

Question 12

What are the substrates for the cell that can be used?What do you co-culture with?

Answer 12

Microcarrier beads, treated plastic, glass, capillaries,collagen coated surface
Feeder cells and stromal cells

Question 13

What are spheroid cells? What’s good about it?

Answer 13

cell cultures that are sphere shaped grwon in viscous scaffold. It mimics the environment found in in vivo, so there are difussion gradients for the cells that are inward and

Question 14

Where do embryonic stem cells come from and what are their characteristics? How is it grown

Answer 14

4-5 days of inner cell mast of embryo.
Indefinitely proliferate and if didn’t differentiate after 6 months are pluripotent and can differentiate into any cell
Grown on feeder cells

Question 15

What are the advantages and disadvantages of cell culture?

Answer 15

Can control physiochemical and physiological factors
Homogenous culture
economical
unlimited supply of cell lines
can store infinitely in liquid nitrogen
Avoids use of animals
-:
expertise, no 3 environment, genetic and phenotypic drift, outgrowth of undifferentiated cells

Question 16

What is confluence?

Answer 16

Reach maximum growth, reach senescence

Question 17

How to produce primary cell culture?

Answer 17

Dissociation-mechanical or enzymes
suspension culture
explant method

Question 18

What factors affect the viability of dissociated cells?

Answer 18

type of animal, tissue, age

concentration of Enzymes used, incubation time, impurities

Question 19

What’s the strongest and the weakest enzyme used?

Answer 19

trypsin and deoxyribonuclease 1

Question 20

What do if low cell yield and low viability?

Answer 20

decrease enzyme concentration

Question 21

what if it’s low cell yield but high viability

Answer 21

increase enzyme concentration

Question 22

high cell yield but low cell viability

Answer 22

decrease enzyme concentration

Question 23

What are cells preserved in? How are they stored

Answer 23

cytoprotective agent such as DMSO( increased serum concentartion allows it to survive)
Liquid nitrogen -196

Question 24

Cells are frozen rapidly and thawed slowly

Answer 24

false, they are frozen slowly and thawed rapidly

Question 25

What are some ways to verify cell lines? Why is it important?

Answer 25

Cytogentic mycoplasma detection DNA/protein profiling To make sure there is no contamination from cell lines or microbes or genetic or phenotype drift

Question 26

How does mycoplasma infection dteection work?

Answer 26

By staining with hoechst stain or PCR or luminescence

Question 27

What induces replicative senescence? What limits the replication? WHy don't cancer cells have this

Answer 27

extrinsic and intrinsic factors TTelomeres shorten witch each division and when it's too short there will be genomic instability and will die. Because their telomerase are active and their p53 or RB might be inactive allowing cells to grow indefinitely

Question 28

What the difference between immortalized and transformed cells?

Answer 28

Immortalized are able to replicate indefinitely through a spontaneous mutation or infection but will not cause cancer if injected in immunocompromised mice and they often gow as a single layer, However transformed cells can cause tumours if injected in mice and have loss of contact inhibition

Question 29

What's transfection? What does the frequency depend on?

Answer 29

``` Insertion of nucleic acid into eukaryotic cells through non viral methods cell number procedure toxicity concentration of nucleic acid ```

Question 30

What are the ways to chemically transfect cells?

Answer 30

DEAE-dextran(positively charged) gets into cell through endocytosis and closely associates with DNA Calcium chloride, DNA and phosphate buffer enter cell through phagocytosis or endocytosis EXGEN500-polymer complexes with DNA and prevents its degradation in lysosome osmotic shock artificial liposomes(doxil)

Question 31

What are physical transfection of the cells?

Answer 31

``` Microinjection Electroporation gene gun magentofection protein transfection(chariot peptide) ```

Question 32

What selection antibody is usually used for transfection?

Answer 32

zeocin

Question 33

What are pHook vectors?

Answer 33

they express single chanin antibodyagainst hapten then magnetic beads coated with hapten select for transfected cells

Question 34

What reporters are used to detect transfected cells?

Answer 34

GFP, luciferase, CAT enzyme activity,bgal, secreted reported protein

Question 35

How are genes quantitaviley regulated?

Answer 35

Tet on and tet off system, genes on in presence of TET but on in the absence of TET. Can be regulated differnetially depending on the concntrations