CRYO#20

Summary

CRYO#20 is a buffer specifically designed for long-term cryogenic preservation of fixed cells processed with the Cytodelics Whole Blood Processing Kit or the PBMC Fixation Kit. It might be used for cryogenic preservation of cells in suspension fixed with other products; however, testing might be required.

For research use only.

Important

DO NOT use for cryogenic preservation of LIVE CELLS.

DO NOT use as an alternative to Cytodelics Stabiliser.

Recommended use

1. CRYO#20 is recommended for cryogenic preservation of fixed leukocytes (isolated from whole blood with the Cytodelics Whole Blood Processing Kit; catalogue numbers hC002-xxxx / mC002-xxxx / WBFL002-xxxx) or PBMCs/cultured cells (PBMCs or other mammalian cell suspensions containing no or a limited number of RBCs, fixed with the Cytodelics PBMC Fixation Kit; catalogue numbers PF002-xxx).
2. The optimal volume per sample/aliquot is 250–500 µL of CRYO#20 for freezing of 1.0–5.0 × 106 cells.
3. Staining with fluorescently labelled antibodies is possible immediately after thawing in CRYO#20 buffer, without the need for washing. However, we recommend testing conditions for each new antibody clone and/or new fluorescent tag.
4. CRYO#20 can be used for cryogenic preservation of cells labelled with fluorescently tagged antibodies.
5. Store CRYO#20 at room temperature.

Protocol 1: Freezing

1. After the last wash of the fixation protocol, aspirate the supernatant, leaving 50–100 µL of the buffer at the bottom of the tube. Break the pellet and bring the fixed cells to suspension.
2. Add 250–500 µL of the CRYO#20 buffer to a suspension of 1–5 × 106 cells; mix well.
3. Transfer to a −80 °C freezer and store. A freezing container is not needed.

Protocol 2: Thawing

1. Set a water bath (or other alternative) to 30–37 °C and wait until the temperature reaches the given interval.
2. Transfer the cryogenically preserved samples to the water bath and thaw quickly within 1–3 minutes. Thawing for more than 5 minutes is not recommended. (Alternative: transfer the sample quantitatively to a centrifugation tube, add PBS (2/3 of the tube’s nominal volume) and spin at 300 g for 5–7 min at room temperature. Aspirate the supernatant.)
3. The sample is ready for further processing (for example staining with an antibody cocktail, or acquisition).

Protocol 3: Thawing and staining without washing

1. Set a water bath (or other alternative) to 30–37 °C and wait until the temperature reaches the given interval.
2. Transfer the cryogenically preserved samples to the water bath and thaw the sample within 1–3 minutes. Thawing for more than 5 minutes is not recommended.
3. Spin at 300 g for 5–7 min at room temperature and aspirate the supernatant, leaving 50–100 µL of the buffer.
4. Add an antibody cocktail and stain either overnight in the fridge, properly sealed to minimise evaporation, or for 30–60 min on ice (if timing is a priority over quality).
5. Wash twice with 2–3 mL of cold PBS. Aspirate carefully to minimise cell loss.
6. Acquire the sample.

Protocol version 1.1, September 2026. The full protocol and the safety data sheet are available under “Support materials” below.