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When to Change

Use this page when splitting/passaging cells and then creating a frozen stock of cells.

Purpose

When passaging cells and a culture reaches confluence, it might be smart to create a frozen stock of cells to keep the cell line alive in case of contamination. Moreover, if a culture is split, there might be too many ongoing flasks so the excess can be used to freeze.

Safety

• Complete work in the BSC.
• Spray down all materials brought into the BSC with 70% Ethanol.
• Try not to touch the sides of the flask opening with pipette tips.
• Do not touch the rims of the flasks or tubes to work with.

Materials

• Cryogenic tubes
• Eppendorf tube
• Cell culture flask
• DMSO
• Complete DMEM

Procedure

*Complete the adhesion removal and suspension steps of Caco-2 Splitting Cells and cell counting steps of Caco-2 Cell Counting

1. Prepare the freezing medium and store at 2-8°C, detatch cells following cell splitting procedure
2. Count cells if need be. When resuspending cells in 3mL of complete DMEM, use 900μL of cells + DMEM and 100μL of DMSO kept in the BSC in a 1.5mL cryogenic tube.
3. Resuspend and mix.
4. Quickly place back into the freezer.

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