Lab 6: Cell Culture

Question 1

What is the required density of HeLa cells per well for transfection?

Answer 1

5 X 10^5 cells per well

This density is aimed to achieve approximately 60-80% confluency.

Question 2

What is the optimal confluency percentage for HeLa cells on the day of transfection?

Answer 2

70% confluency

Achieving this confluency is necessary for optimum transfection efficiency.

Question 3

What medium is used to maintain HeLa cells?

Answer 3

RPMI containing 10% new born calf serum

This medium supports the growth and maintenance of HeLa cells.

Question 4

How many HeLa cells are needed to achieve the required confluency in a 35mm dish after 24 hours?

Answer 4

2-3 X 10^5 HeLa cells

This range ensures the cells reach the appropriate confluency for transfection.

Question 5

What is the first step in the trypsinisation protocol?

Answer 5

Pour medium from the flask into a sterile universal container

This step is necessary to inactivate trypsin later in the process.

Question 6

How should the cells be washed before trypsinisation?

Answer 6

Twice with sterile PBS

PBS washes help to remove excess serum and prepare the cells for trypsin treatment.

Question 7

What should be done after adding trypsin to the flask?

Answer 7

Swirl to cover cells and pour most out immediately

This ensures that the trypsin can effectively detach the cells.

Question 8

What indicates that the cells are ready to be detached during trypsinisation?

Answer 8

Cells begin to round up and detach

This observation can be made using an inverted microscope.

Question 9

What is the centrifugation speed and time for pelleting the cells?

Answer 9

1500 rpm for 7 minutes

This speed and duration are optimal for pelleting the HeLa cells after trypsinisation.

Question 10

How should the cell suspension be prepared for counting?

Answer 10

Resuspend cells in 2ml of PBS and mix with trypan blue

40 μl of cell suspension should be combined with 40 μl of trypan blue.

Question 11

What is the purpose of trypan blue in cell counting?

Answer 11

To distinguish viable cells from non-viable cells

Viable cells exclude the dye, while non-viable cells take it up.

Question 12

How is the final cell concentration calculated after counting?

Answer 12

Multiply cell number by 2 (trypan blue dilution) and by 10

This calculation gives the number of cells per ml.

Question 13

Fill in the blank: Cells should be incubated at _______ overnight.

Answer 13

37°C (5% CO2)

This incubation condition is crucial for optimal cell growth.

Question 14

True or False: HeLa cells need to be 60-80% confluent before transfection.

Answer 14

True

This range is necessary to ensure effective transfection.

Question 15

What is the purpose of the pCMV sport B gal vector?

Answer 15

It drives the expression of the ß galactosidase gene under the control of the CMV enhancer

This vector is used in transfection protocols to study gene expression.

Question 16

What is the function of the pCH110 vector?

Answer 16

It drives the expression of the ß galactosidase gene under the control of the SV40 enhancer

This vector is also used in transfection protocols to facilitate gene expression studies.

Question 17

How many wells are available for transfection in the protocol?

Answer 17

5 wells

Out of these, 2 wells remain untransfected as controls.

Question 18

What is the first step in the transfection protocol?

Answer 18

Add 100 µl of warm RPMI into an Eppendorf tube

This is followed by adding the transfection reagent.

Question 19

What is the incubation time after adding the plasmid DNA to the transfection mix?

Answer 19

10 minutes at room temperature

This step is crucial for allowing the DNA to interact with the transfection reagent.

Question 20

What is the purpose of the in situ staining of HEP2 cells?

Answer 20

To detect B-galactosidase activity in the cells

This staining helps visualize the expression of the transfected gene.

Question 21

What should be done after aspirating the cell culture medium?

Answer 21

Wash the cells twice carefully with 2 ml of PBS

It is important to ensure that all PBS is removed after the second wash.

Question 22

Fill in the blank: The transfection reagent is added at a volume of _______.

Answer 22

3 µl

This reagent is critical for facilitating the uptake of plasmid DNA by the cells.

Question 23

True or False: Cells transfected with pCMV sport B gal can only be detected in the cell extract.

Answer 23

False

B-galactosidase can be detected both in the cell extract and in situ.

Question 24

What is the incubation temperature for the X-gal solution during staining?

Answer 24

37°C

This temperature is optimal for the enzymatic reaction to occur.

Question 25

What should be used to fix cells after washing?

Answer 25

1 ml of glutaraldehyde solution

Fixation is essential for preserving cell morphology and enzyme activity.

Question 26

What is the duration for fixing cells with glutaraldehyde?

Answer 26

15 minutes

This allows adequate fixation of the cells before further processing.

Question 27

How many times should cells be washed after fixing with glutaraldehyde?

Answer 27

3 times

This washing step is necessary to remove excess fixative.

Question 28

What is the final step in the transfection protocol?

Answer 28

Incubate in the cell culture incubator for 48 hours

This period allows for the expression of the transfected genes.

Question 29

What is the purpose of preparing extracts from cells transfected with pCHI10 and pCMV sport ß gal?

Answer 29

To assay for B-galactosidase activity in a liquid assay

This process helps in evaluating the expression of the transfected genes.

Question 30

What is the first step in the procedure for preparing cell extracts?

Answer 30

Aspirate medium from the plates

This step is crucial to remove any residual culture medium before washing the cells.

Question 31

How many times should cells be washed with PBS during the preparation process?

Answer 31

Twice

This ensures that the cells are clean and free from serum components that might interfere with the assay.

Question 32

What volume of lysis buffer is added to the plate?

Answer 32

200ul

The lysis buffer helps to break open the cells to release their contents for the assay.

Question 33

For how long should the cells be incubated with lysis buffer at room temperature?

Answer 33

15 minutes

Periodic gentle rocking during this time helps to ensure complete lysis of the cells.

Question 34

What is done to the cells after incubation with lysis buffer?

Answer 34

Scrape cells from the plate and transfer the lysate to an eppendorf tube

This step collects the cell lysate for further processing.

Question 35

What is the purpose of vortexing and spinning the lysate at maximum speed?

Answer 35

To ensure complete mixing and to separate the supernatant from the cell debris

This is important for obtaining a clear extract for the assay.

Question 36

What components are included in the 2X assay buffer?

Answer 36

• 200mM sodium phosphate buffer, pH 7.3
• 2mM MgCl₂
• 100mM β-ME
• 1.33mg/ml ONPG

These components are essential for the enzymatic reaction to occur during the assay.

Question 37

What should be done after adding the cell extract and 2X assay buffer together?

Answer 37

Incubate at 37°C until a faint yellow colour develops

The development of color indicates the activity of B-galactosidase.

Question 38

What is the next step after incubating the mixture until a faint yellow color develops?

Answer 38

Add 500ul of 1M sodium carbonate to stop the reaction

Stopping the reaction is necessary to measure the absorbance accurately.

Question 39

At what wavelength should the absorbance be read after the reaction is stopped?

Answer 39

420 nm

This wavelength is optimal for detecting the product of the B-galactosidase reaction.