Our lab has some radioactive cell samples that need to be kept frozen for at least 30 days before flow analysis. For some reason, the samples stored in liquid nitrogen did not perform as well as others. For example, the CD45 positive population shifted from 10^3 to 10^2 or even lower in Flowjo. And it is though to distinguish the positive population from the negative part.
Is that normal, and has anyone solved that?
Storing radioactive cell samples in liquid nitrogen for extended periods can sometimes lead to changes in cell surface marker expression, which might explain the shift in the CD45 positive population you observed. Here are some possible reasons for this phenomenon and suggestions to mitigate it:
### Potential Issues
#### 1. **Cryopreservation Effects**
- **Cell Membrane Integrity**: The extreme cold of liquid nitrogen can sometimes cause damage to cell membranes, affecting the binding of antibodies to cell surface markers.
- **Ice Crystal Formation**: Improper freezing rates can lead to the formation of ice crystals, which can physically damage cells and alter the expression of surface markers.
#### 2. **Freezing and Thawing Process**
- **Thawing Method**: Rapid or uneven thawing can cause cell stress and affect the integrity of surface markers. It's crucial to follow a consistent and controlled thawing protocol.
- **Cryoprotectant Use**: Ensure the appropriate concentration of cryoprotectants like DMSO is used. Incorrect concentrations can either be insufficient to prevent ice formation or toxic to cells.
#### 3. **Radioactivity Effects**
- **Radiation Damage**: Prolonged exposure to radiation, even at low levels, can cause cellular stress and alter cell surface proteins. Ensure that the level of radioactivity is within safe limits for the cells.
### Best Practices for Cryopreservation
1. **Optimized Cryoprotectant Protocol**
- Use 10% DMSO in combination with a suitable medium (e.g., FBS or a cryopreservation medium) to protect cells during freezing.
- Aliquot cells into cryovials at a consistent cell concentration (e.g., 1-5 million cells per mL).
2. **Controlled Freezing Rate**
- Use a controlled-rate freezer to gradually cool the samples to -80°C before transferring them to liquid nitrogen. A typical rate is -1°C per minute.
3. **Thawing Protocol**
- Thaw the cells rapidly by placing the cryovial in a 37°C water bath. Once thawed, immediately dilute the cells in pre-warmed medium to reduce the concentration of DMSO.
- Gently mix and then centrifuge to remove the cryoprotectant.
4. **Handling Radioactive Samples**
- Store and handle radioactive samples following all safety protocols to minimize any potential radiation damage to the cells.
- If possible, reduce the storage time of radioactive samples in liquid nitrogen to the minimum required.
### Recommendations for Flow Cytometry Analysis
- **Staining Procedure**: Ensure that the staining protocol is optimized for cryopreserved cells. Sometimes, slight modifications are needed compared to fresh cells.
- **Compensation and Controls**: Use proper compensation controls and unstained controls to accurately set gates and distinguish between positive and negative populations.
### Literature and Protocols
1. **Cryopreservation of Cells**:
- Mazur, P. (1963). "Cryobiology: the freezing of biological systems." Science, 138(3544), 1271-1279.
- Stacey, G. N., & Hartung, S. (2006). "Cryopreservation and banking of human ES cells." Nature Protocols, 1(1), 212-218.
2. **Flow Cytometry on Frozen Samples**:
- Perfetto, S. P., Ambrozak, D., Nguyen, R., & Roederer, M. (2010). "Quality assurance for polychromatic flow cytometry." Nature Protocols, 5(2), 445-456.
By following these best practices and protocols, you should be able to improve the viability and performance of your radioactive cell samples during cryopreservation and subsequent flow cytometry analysis.
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