Passaging Cells: Cell Culture Basics

Summary

This video script outlines the proper procedure for maintaining and passaging cultured cells. It covers key phases, including the log phase of growth, sterilizing equipment, cell detachment using dissociation reagents, centrifugation, and cell counting with Trypan Blue. The script emphasizes the importance of using gentle methods to avoid cell damage, as well as ensuring single-cell suspension for accurate counts. Additionally, it highlights maintaining proper medium levels, using correct flask caps for ventilation, and routine culture maintenance, such as replacing medium every three weeks to avoid cell debris buildup and metabolic waste.

Takeaways

• 😀 Cells in culture follow a growth pattern, with a log phase of exponential growth after seeding.
• 😀 Cells should be passaged when they cover the plate or exceed the medium's capacity.
• 😀 Maintain log phase growth to maximize healthy cell numbers for experiments.
• 😀 Before retrieving flasks from the incubator, sterilize the hood and gather necessary supplies.
• 😀 Carefully examine cultures for signs of contamination or deterioration before handling.
• 😀 For adherent cells, rinse with a balanced salt solution like DPPS before dissociation.
• 😀 Use a cell dissociation reagent to detach cells, ensuring it covers the entire cell sheet.
• 😀 Confirm complete dissociation by using a microscope to observe the cells detaching and becoming round.
• 😀 After dissociation, centrifuge the cells, discard the medium, and resuspend the pellet in warm complete growth medium.
• 😀 Perform a cell count using trypan blue to assess cell viability by identifying live and dead cells.
• 😀 Based on the cell count, adjust medium volume for optimal seeding density, and distribute cells evenly into fresh flasks.

Q & A

What is the first step after seeding cells in culture?

The first step is a lag phase followed by a period of exponential growth known as the log phase.

When should cells be passaged?

Cells should be passaged when they cover the plate or when the cell density exceeds the capacity of the medium.

Why is it important to maintain log phase growth?

Maintaining log phase growth maximizes the number of healthy cells for your experiment.

What should you do before retrieving flasks from the incubator?

Before retrieving flasks, sterilize the hood and ensure all required supplies are at hand.

How can you ensure that the cells are not contaminated?

Carefully examine the culture for signs of contamination or deterioration before handling.

What is the role of the salt solution when preparing adherent cells?

The salt solution, such as DPPS, is used to rinse the cells before adding the dissociation reagent.

Why should a calcium and magnesium-free solution be used?

A calcium and magnesium-free solution is important because these ions may inhibit the action of the cell dissociation reagent.

What are the signs that cells have successfully detached from the culture surface?

Cells will appear round and will move or slide when the flask is tilted, indicating they have detached from the substrate.

What should you do if cells are not fully dissociated after applying the reagent?

If cells are not fully dissociated, gently pipette warmed medium over any lingering clumps to break them apart.

What is the purpose of using Trypan Blue in cell counting?

Trypan Blue is used to distinguish dead cells, as it stains dead cells blue, while live cells remain colorless.