high yield basics of immu3102

Question 1

What is in the cell culture media

Answer 1

DMEM: Dulbecco’s modified Eagle medium

Question 2

What is in the culture medium supplment and what are the conditions of the incubation

Answer 2

essential amino acids, feta calf serum, antibiotics and phenol red.

37 C cell culture incubator with 5% CO2

Question 3

What is the point of the Contaminated air negative pressure

Answer 3

It prevents contaminated air from coming into the cabinet

Question 4

(What is the point of the HEPA filtered air) answer actually unclear

Answer 4

to prevent potentially contaminated air coming back out to the operator.

Question 5

What TLR recognises LPS

Answer 5

TLR4

Question 6

What does the TLR recognition of microbial ligands do?

Answer 6

It activates signalling pathways,
transcription factors
and the expression of genes important in inflammatory and antiviral responses

Question 7

How do we detect protein in culture supernatants?

Answer 7

ELISA

Question 8

How do we detect protein in cell lysates

Answer 8

western blottin

Question 9

How do we detect mRNA in cell lysates

Answer 9

real time PCR

Question 10

how do we detect protein in fixed cells

Answer 10

immunostaining

Question 11

How do we detect mRNA in fixed cells?

Answer 11

FISH

Question 12

describe the process of NFkB activation

Answer 12

IKK is made of IKK a, b and gamma

IkB a is basically degraded, leaving p65 and p50 then NFkB is imported to a nucleus.

Question 13

What does PFA do

Answer 13

paraformaldehyde terminates reaction and preserve morphology

Question 14

How do you permeabilize cells

Answer 14

Triton X-100

Question 15

What is the primary antibody used to measure NFkB translocation

Answer 15

rabbit anti-human NFkB p65

Question 16

What is the secondary antibody used in NFkB translocation

Answer 16

AF488 conjugated goat antirabbit IgG

Question 17

What is the function of DAPI

Answer 17

it is a nuclear counterstain (blue-fluorescent 4, 6 diamidino 2 phenylindole)

Question 18

What do cells look like when they have no stimulus

Answer 18

The outside is green due to NFkB, and there is DAPI in the centre.

When there is LPS, they centre of cells become cyan
as green+blue=cyan

Question 19

What is the percentage of B cells in the spleen?

Answer 19

50%

Question 20

What is the percentage of CD4 T cells in the spleen

Answer 20

20%

Question 21

What is the percentage of CD8 T cells in the spleen?

Answer 21

10%

Question 22

What is the percentage of other kinds of immune cells in the spleen?

Answer 22

20%

Question 23

How many molecules of biotin does streptavidin bind to

Answer 23

Streptavidin is a protein produced by the bacteria Streptomyces avidinii,
Usually 1 molecule of streptavidin could bind to 4 molecules of biotin

Question 24

Why do we use biotin and streptavidin in separating immune cells

Answer 24

Because this interaction is resistant to detergents, enzymes, solvents and extremes in pH and temperature.

Question 25

How does the biotin and streptavidin interaction work?

Answer 25

basically you have a magnetic bead coated with streptavidin. the streptavidin would bind with the affinity bound immobilised antibody. And then the cells are captured on that antibody

Question 26

What are we pouring off into a new tube

Answer 26

The biotinylated antibodies are specific for markers not expressed on CD4. So, the magnet attracts any non-CD4 cells.

We pour off the desired CD4 T cells

Question 27

how does specific antibody mix work

Answer 27

anti CD3-FITC binds to CD3 on all mouse T cells
anti-B220 PerCP binds to B220 on all mouse B cells
anti CD4PE binds to CD4 on mouse T cells
anti CD 8 APC binds to any CD8