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Shanghai Jinma Bio has developed a practical method for the preservation and transportation of cells commonly used by our customers. We have posted this information on our website for your reference. If you have any suggestions or feedback regarding the methods we provide, please feel free to contact us. Our secretary is available to take your call — we look forward to hearing from you.
After preparing the cell suspension, Shanghai Jinma calculates the number of cells present, typically expressed as cells per milliliter (cells/ml). This process ensures accurate tracking and handling of cell cultures.
**Materials Required:** - Cell suspension - Counting plate (hemocytometer) - Microscope - Pipette
**Procedure:** 1. **Preparation:** - Rinse the counting plate with alcohol, wipe it dry, and place the cover slip over the counting grid. Slightly lift the cover slip to add the cell suspension dropwise along the edge. - Avoid air bubbles and ensure even distribution without overflowing. 2. **Counting:** - Under the microscope, count the cells in the four corner squares of the counting plate. Only count cells that are on the top and left lines; exclude those on the right and bottom lines to avoid double-counting. 3. **Calculation:** - The formula used is: **Cell number / ml = (Number of cells counted × dilution factor) / volume of the counting area** - For example: If you count 110 cells in one square, and the dilution factor is 10, then the final concentration would be 1100 cells/ml.
**Notes:** - If clusters of cells are observed, they should be counted as single units. If more than 10% of the cells appear in clusters, it may indicate incomplete digestion. - If the cell count is less than 200 or more than 500 cells per 10 mm², the dilution may be incorrect. In such cases, re-prepare the cell suspension and recount.
**Cryopreservation of Cultured Cells** Adding a cryoprotectant such as dimethyl sulfoxide (DMSO) to the culture medium helps protect cells from damage caused by ice crystal formation and osmotic stress during freezing. With proper techniques, cells can be stored in liquid nitrogen at -190°C for long-term preservation.
**Materials Needed:** - Cryopreservation tube - DMSO or glycerol (cryoprotectant) - Culture medium - Fetal bovine serum (FBS) - Liquid nitrogen tank
**Steps for Cryopreservation:** 1. Collect cells during the logarithmic growth phase. 2. Mix the cells with a cryopreservation solution containing 10–20% FBS and 5–10% DMSO. 3. Adjust the cell concentration to approximately 1–2 million cells/mL. 4. Transfer the mixture into a cryotube and label it clearly. 5. Freeze the cells gradually at a rate of 1–10°C/min. 6. If no controlled-rate freezer is available, place the tube in a 4°C refrigerator for 4 hours before transferring to liquid nitrogen. 7. Store the tube in the gas phase of the liquid nitrogen container for 1 hour before submerging it in the liquid nitrogen.
**Cell Recovery Procedure** To revive cryopreserved cells, follow these steps: 1. Remove the cryotube from the liquid nitrogen. 2. Immerse it in a water bath at 38°C for 20–60 seconds, shaking gently until fully melted. 3. Open the tube aseptically in a laminar flow hood. 4. Transfer the cells into a culture flask and add fresh culture medium. 5. Incubate at 37°C for 24 hours. 6. Replace the medium to remove the cryoprotectant and continue the culture.
**Transportation of Cultured Cells** For short-term transport, cells can be shipped in sealed culture flasks filled with fresh media. This method is convenient and widely used. **Steps for Transport:** 1. Select healthy, well-grown cells. 2. After reaching near confluence, remove the old media and replace it with fresh media. 3. Fill the flask up to the neck, secure the lid, and leave a small amount of air inside. 4. Avoid excessive air to prevent bubble formation during transit. 5. Cells can typically survive for 4–5 days under normal conditions. Prolonged transport may reduce viability. **Upon Arrival:** 1. Pour out most of the media, leaving just enough to sustain the cells. 2. Incubate at 37°C and proceed with the next passage the following day.
We hope this detailed guide helps in your cell culture and transport processes. Should you need further assistance, don’t hesitate to reach out.
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