immu3102 practical

Question 1

What are a set of basic environmental requirements for cells to grow in cell culture

Answer 1

controlled temperature (37 C for mammalian cell culture)

Appropriate growth medium

Appropriate pH and osmolality

Question 2

What should a cell culture medium contain?

Answer 2

source of energy (usually glucose)
aminoacids
vitamins
inorganic salts.

Question 3

How is pH kept constant in a cell culture medium

Answer 3

The use of a natural buffering system where the incubator balances with the CO3/HCO# contained in the cell culture medium

Question 4

Outline how phenol red works

Answer 4

It is an indicator
It is red at optimal pH
It turns pink/purple at high pH
And it turns yellow at low pH

Question 5

Aseptic technique

Answer 5

ethanol
don't clutter
don't touch tip of any containers or pipettes
place cap on its side.
Common sense really. 

Perform all procedures in a biological safety cabinet and wear gloves and lab coat throughout the procedures.
Wipe all surfaces before use by swabbing down with 80% ethanol. NB: Just because you have swabbed with 80% ethanol this does not mean the surfaces or your gloves are sterile.
Periodically cover exterior of gloves with 80% ethanol to minimise contamination. Replace gloves if torn.
Only have items that are needed for the procedure in the biological safety cabinet.
Leave a wide clear space in the centre of the cabinet to work on. Do not clutter the cabinet as it may prevent proper air flow and may cause turbulence.
Arrange the work area to have easy access to all items without having to reach over one item to get to another (especially over an open bottle or flask).
All equipment to be used must be sterile (autoclaved, gamma irradiated or filter sterilized). Inspect items to be used for broken or damaged wrapping.
Use sterile wrapped pipettes, never pipette by mouth; always use an automatic pipetting device.
Make sure not to touch the tip of the pipette to the rim of any flask or bottle.
Be careful when removing sterile items from wrappings to prevent them touching any surface including your gloves.
When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
Discard disposable equipment into a biohazard waste container or autoclave bag immediately after use. Do not leave waste in the cabinet.
Clean the work area and when finished wipe with 80% ethanol.

Question 6

How is a fungal contamination identified

Answer 6

There is a large fungal mass floating in the bottle of cell culture media that you have been using

Question 7

How is bacterial contamination identified macroscopically

Answer 7

usually the culture media becomes very cloudy. Bacteria and their acidic metabolic by-products may also cause the media in your cell culture to become acidic

Question 8

How is bacterial contamination of a cell observed microscopically

Answer 8

Fuzzy brownian motion of bacteria.

Question 9

what are 2 ways apart from the aseptic technique that could help control contamination

Answer 9

use antibiotics

take extreme care with your waste bottle

being careful in water baths

Question 10

How do you take care using a water bath

Answer 10

Water baths are a common source of contamination as water is not changed before every use

never fully immerse a vial of cells
do not dip the vial into the water deep enough to submerge any part of the lid as this may allow water to get in under the lid

Thoroughly sterilise the outside of the tube with 80% v/v ethanol before transferring the vial into the biosafety cabinet

Question 11

growing ARPE cells

Answer 11

?

Question 12

What are the size of the squares for the haemocytometer

Answer 12

1.0mm on each side
The cover class is supported 0.1 mm over these squares

Question 13

Why must you shake the cell suspension and mix it thoroughly before you shake it

Answer 13

It is assumed that the total volume in the chamber represents a random sample. And this will not be a valid assumption unless the suspension consists of individual separated cells. Unless 90% or more of the cells are free from contact with other cells the count should be repeated with a new sample.

Question 14

How many cells should be counted in a haemocytometer

Answer 14

200-300, each square should have 30-70 cells

Question 15

Counting convention for the haemocytometer

Answer 15

top and left borders included. Bottom and right borders included

Question 16

What is the point of Trypan Blue, nigrosine and eosin

Answer 16

These are taken up in dead cells in a haemocytometer, but excluded in live cells.

Question 17

What is the concentration of cells in the original suspension

Answer 17

Nx10^4 x dilution factor/ Number of 1mm squares counted

Question 18

What is the primary antibody we are using for our immunofluorescent assay

Answer 18

A rabbit antibody against NF-kB p65

Question 19

What is the secondary antibody

Answer 19

fluorescently labelled secondary antibody raised in goat

Question 20

What does the secondary antibody do

Answer 20

It is an antibody which is conjugated to a fluorescent marker and emits light when excited at the correct wave length.

Question 21

What is the difference between immunohistochemistry and immunofluorescence

Answer 21

Immunohistochemistry use secondary antibodies that are conjugated to enzymes and produce a coloured precipitate upon addition of a substrate

Question 22

What is the point of a counterstain in the immunofluorescent assay

Answer 22

It helps us visualise the structure of the cells under the microscope. It allows us to assess the staining pattern, the localisation of the stain within the cells

Question 23

What is a chamber slide

Answer 23

a glass slide that has a plastic divider adhered to it that divides the slide into wells. Cells can be grown in the wells and have different treatments applied without having solutions from one well crossing into another. The chamber from the slide leaves separate sections of cells for subsequent immunostainin

Question 24

What is a humidified chamber

Answer 24

basically a rack to place the slides on

Wet tissues or paper towels create the humid environment a lid to keep the humidity in the chamber.

Question 25

Why is a humidified chamber required

Answer 25

The small volume of antibody added to each section would evaporate if it was not contained in a humidified chamber. Evaporation is bad for immunostaining as it can result in significant non-specific staining

Question 26

What is the point of the mounting medium

Answer 26

typically contains an “anti-fade” reagent that preserves the fluorescence in the sample and also a nuclear counterstain such as DAPI. The use of an anti-fade reagent means that you have longer to view and photograph your experiment before it is no longer detectable

Question 27

In our ELISA experiment, what were we trying to test the presence for?

Answer 27

TNF alpha

Question 28

How does a cytokine ELISA work?

Answer 28

First coat the ELISA plate with a cytokine specific capture antibody then add your sample which contains the cytokine of interest. To detect and visualise the bound cytokine, the anti-cytokine antibody conjugated to an enzyme is then added.

Question 29

What is the role of alkaline phosphatase

Answer 29

it is an enzyme that is conjugated to our second anti-cytokine antibody. We could add our substrate, p-nitrophenyl phosphate to produce a yellow colour change

Question 30

What is facswash and what does it do?

Answer 30

Basically it is a dilute solution of protein in PBS.

1) improves cell viability
2) reduces clumping of cells
3) blocks non-specific stain
4) prevents heterophilic antibody interference.

Question 31

How do you separate lymphocytes from a mixed suspension

Answer 31

Using antibody coated magnetic beads and magnetic separation approach. After the antibodies have bound to their corresponding cell surface antigens, the beads and the cells attached to the beads are separated from cell suspension by resting in a very strong magnetic field

Question 32

What is the isotype control mix

Answer 32

It is a mix of 4 antibodies conjugated with four fluorochromes. The antibodies are from the same species and have the same isotype as those in the antibody mix, but they do not bind mouse lymphocytes

Question 33

Why is an isotype control mix added?

Answer 33

So, isotype control is the same type of antibody as the antibody that recognises your antigen of interest but can’t bind.

If the cells in your experiment had an affinity for the particular antibody isotype regardless of what antibody it is, then your specific antibody and your isotype control would both bind to the tissue or cells.

Isotype controls are important as they are a negative control that helps to differentiate non-specific background signal from specific antibody signal, confirms any signal observed with your specific antibody is true positive signal