Adherent cell subculture

Question 1

Steps to follow for appropriate lab technique

Answer 1

1. Lab workers have to prepare themselves using aseptic technique and good personal hygiene
2. Prepare the reagents by putting it into an incubator or water bath
3. The reagents have to be sprayed with disinfectant before adding them to the hood
4. Work area has to be cleaned by spraying with 70% ethanol and disinfectant

Question 2

Ways to organise the workspace

Answer 2

Prevents disrupting the airflow from making sure nothing is on the grill

Lab workers gather everything they need to prevent moving around

Workers should organise the area around their workspace

Question 3

Apparatus used in the work space

Answer 3

Serological pipettes

Tissue to wipe or clean spillage

Waste container

Tube rack

Culture plasticware

Pipettor

Question 4

What is PBS used for?

Answer 4

Phosphate buffer saline

Wash cells before the addition of the dissociation agent

Question 5

Important aspect of waste container

Answer 5

Supplemented with disinfectant agent

Question 6

How do we check cell viability?

Answer 6

Check colour/clearness of medium

Cultures should be observed daily in order to become familiar with the morphological appearance of your cell line

Question 7

Morphological signs of deterioration

Answer 7

Pignotic aspects of the nucleus

Detatchment of cells from substrate

Vacuolation in cytoplasm

Question 8

Causes of cell deterioration

Answer 8

Contamination

Cell senescence

Needs medium change

Question 9

What is the normal amount of time cell stocks are used in culture?

Answer 9

3 months

Question 10

What is regularly tested in cell culture?

Answer 10

Mycoplasma infection

If infected - discard or treat with plasmocin

Question 11

When do you have to split your cells?

Answer 11

When the culture reaches 80-90% confluency

Question 12

What does the time required for trypsinisation to occur depend on?

Answer 12

The cell type

Confluency

Concentration of trypsin used

Question 13

What is a sign a cell has trypsinised?

Answer 13

Cell morphology changes from pellet-like to round

Cells move since they no longer adhere to plastic

Question 14

How is trypsin deactivated?

Answer 14

Adding Ca2+ or Mg2+

Question 15

How to pellet cells from a cell culture

Answer 15

Suspended cells in a total volume of 10-15ml are placed in a 25ml falcon tube

Spun down at 1000g for 10 minutes

Question 16

How to label cell culture flask

Answer 16

Your name

Cell line name

Number of passages

Other necessary details - drug treatment, serum presence

Question 17

How is subculturing personalised?

Answer 17

The split you do depends on the type of cell you have

Delicate or slow-growing cells = 1:2 split
Cancer or immortalized cell line = 1:5 or 1:10 split

Question 18

What are two apparatus used to count cells?

Answer 18

Automated cell counter

Haemocytometer

Question 19

How to count cells using a haemocytometer

Answer 19

Using a pipette, take 100 µL of Trypan Blue-treated cell suspension and apply to the hemocytometer.

Using a microscope, focus on the grid lines of the hemocytometer with a 10X objective.

Using a hand tally counter, count the live, unstained cells

Move the hemocytometer to the next set of 16 corner squares and carry on counting until all 4 sets of 16 corners are counted

Question 20

How to calculate the number of viable cells

Answer 20

Take the average cell count from each of the sets of 16 corner squares.

Multiply by 10,000 (10^4).

Multiply by 5 to correct for the 1:5 dilution from the Trypan Blue addition.

Question 21

Is overheating or underheating cells more of a problem?

Answer 21

Overheating

Question 22

Why is carbon dioxide important to control in cell culture?

Answer 22

Changes the pH of the medium

Question 23

What is the normal range of carbon dioxide used in cell culture?

Answer 23

5-7%

Recommended to check the properties of your medium and the manufacturer’s recommendation

Question 24

How do you clean an incubator?

Answer 24

Using Virkon or detergent + 70% ethanol

Some have decontamination mode - temperature of incubatory is raised to 180 degrees