high yield basics of immu3102 Flashcards
What is in the cell culture media
DMEM: Dulbecco’s modified Eagle medium
What is in the culture medium supplment and what are the conditions of the incubation
essential amino acids, feta calf serum, antibiotics and phenol red.
37 C cell culture incubator with 5% CO2
What is the point of the Contaminated air negative pressure
It prevents contaminated air from coming into the cabinet
(What is the point of the HEPA filtered air) answer actually unclear
to prevent potentially contaminated air coming back out to the operator.
What TLR recognises LPS
TLR4
What does the TLR recognition of microbial ligands do?
It activates signalling pathways,
transcription factors
and the expression of genes important in inflammatory and antiviral responses
How do we detect protein in culture supernatants?
ELISA
How do we detect protein in cell lysates
western blottin
How do we detect mRNA in cell lysates
real time PCR
how do we detect protein in fixed cells
immunostaining
How do we detect mRNA in fixed cells?
FISH
describe the process of NFkB activation
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.
What does PFA do
paraformaldehyde terminates reaction and preserve morphology
How do you permeabilize cells
Triton X-100
What is the primary antibody used to measure NFkB translocation
rabbit anti-human NFkB p65
What is the secondary antibody used in NFkB translocation
AF488 conjugated goat antirabbit IgG
What is the function of DAPI
it is a nuclear counterstain (blue-fluorescent 4, 6 diamidino 2 phenylindole)
What do cells look like when they have no stimulus
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
What is the percentage of B cells in the spleen?
50%
What is the percentage of CD4 T cells in the spleen
20%
What is the percentage of CD8 T cells in the spleen?
10%
What is the percentage of other kinds of immune cells in the spleen?
20%
How many molecules of biotin does streptavidin bind to
Streptavidin is a protein produced by the bacteria Streptomyces avidinii,
Usually 1 molecule of streptavidin could bind to 4 molecules of biotin
Why do we use biotin and streptavidin in separating immune cells
Because this interaction is resistant to detergents, enzymes, solvents and extremes in pH and temperature.
