Adherent cell subculture Flashcards
Steps to follow for appropriate lab technique
- Lab workers have to prepare themselves using aseptic technique and good personal hygiene
- Prepare the reagents by putting it into an incubator or water bath
- The reagents have to be sprayed with disinfectant before adding them to the hood
- Work area has to be cleaned by spraying with 70% ethanol and disinfectant
Ways to organise the workspace
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
Apparatus used in the work space
Serological pipettes
Tissue to wipe or clean spillage
Waste container
Tube rack
Culture plasticware
Pipettor
What is PBS used for?
Phosphate buffer saline
Wash cells before the addition of the dissociation agent
Important aspect of waste container
Supplemented with disinfectant agent
How do we check cell viability?
Check colour/clearness of medium
Cultures should be observed daily in order to become familiar with the morphological appearance of your cell line
Morphological signs of deterioration
Pignotic aspects of the nucleus
Detatchment of cells from substrate
Vacuolation in cytoplasm
Causes of cell deterioration
Contamination
Cell senescence
Needs medium change
What is the normal amount of time cell stocks are used in culture?
3 months
What is regularly tested in cell culture?
Mycoplasma infection
If infected - discard or treat with plasmocin
When do you have to split your cells?
When the culture reaches 80-90% confluency
What does the time required for trypsinisation to occur depend on?
The cell type
Confluency
Concentration of trypsin used
What is a sign a cell has trypsinised?
Cell morphology changes from pellet-like to round
Cells move since they no longer adhere to plastic
How is trypsin deactivated?
Adding Ca2+ or Mg2+
How to pellet cells from a cell culture
Suspended cells in a total volume of 10-15ml are placed in a 25ml falcon tube
Spun down at 1000g for 10 minutes
How to label cell culture flask
Your name
Cell line name
Number of passages
Other necessary details - drug treatment, serum presence
How is subculturing personalised?
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
What are two apparatus used to count cells?
Automated cell counter
Haemocytometer
How to count cells using a haemocytometer
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
How to calculate the number of viable cells
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.
Is overheating or underheating cells more of a problem?
Overheating
Why is carbon dioxide important to control in cell culture?
Changes the pH of the medium
What is the normal range of carbon dioxide used in cell culture?
5-7%
Recommended to check the properties of your medium and the manufacturer’s recommendation
How do you clean an incubator?
Using Virkon or detergent + 70% ethanol
Some have decontamination mode - temperature of incubatory is raised to 180 degrees
