59M

Frequently Asked Questions

• What is the recommended seeding density for 59M cells?

  The recommended seeding density for 59M cells can vary depending on the specific experimental needs and growth characteristics. As a general guideline:

  Initial Seeding Density: Start with a seeding density of around 5,000 to 10,000 cells per square centimeter (cells/cm²) of growth area. Adjust based on the growth rate and experimental requirements.

• What is the recommended culture medium and conditions for 59M cells?

  Recommended Culture Medium and Conditions for 59M Cells:
  Culture Medium: 59M cells are typically cultured in RPMI-1640 medium supplemented with fetal bovine serum (FBS), usually at a concentration of 10% to 20%. Antibiotics such as penicillin-streptomycin are commonly added to prevent contamination.

  Temperature and Atmosphere: Maintain the cells at 37°C in a humidified atmosphere containing 5% CO₂. This environment helps to mimic physiological conditions and supports cell growth.

• What are common issues encountered with 59M cell culture and how can I troubleshoot them?

  Poor Cell Attachment: Ensure that culture vessels are properly coated with extracellular matrix proteins (e.g., collagen, fibronectin) before seeding. Also, avoid over-trypsinization during passaging, as excessive trypsin can damage cell membranes and affect attachment.

  Slow Growth or Reduced Viability: Check the freshness and quality of the medium and FBS used. Serum should be heat-inactivated to minimize the risk of viral contamination. Ensure that cells are not over-confluent and are regularly subcultured to maintain active growth.

  Contamination: Maintain strict aseptic techniques, including frequent cleaning of the work area and equipment. Use antibiotics judiciously to prevent bacterial or fungal contamination.

  pH Imbalance: Monitor the pH of the medium regularly and adjust if necessary to maintain optimal conditions for cell growth (typically pH 7.2-7.4).

• How do I cryopreserve and thaw 59M cells while maintaining their viability and functionality?

  Cryopreservation and Thawing of 59M Cells:
  Cryopreservation:

  Preparation: Use a freezing medium containing RPMI-1640 supplemented with 10-20% FBS and 10% dimethyl sulfoxide (DMSO). DMSO helps prevent ice crystal formation during freezing, which can damage cells.

  Freezing Protocol:

  Harvest cells during exponential growth phase.
  Resuspend cells in the freezing medium at a concentration of 1-2 million cells per mL.
  Transfer aliquots of the cell suspension to cryovials.
  Place cryovials in a controlled-rate freezer or a -80°C freezer for slow freezing.
  Transfer cryovials to liquid nitrogen for long-term storage (below -130°C).
  Thawing:

  Thawing Protocol:
  Quickly thaw cryovials in a 37°C water bath until only a small ice crystal remains.
  Transfer the cell suspension to a tube containing pre-warmed RPMI-1640 medium with FBS (10-20%).
  Centrifuge cells at low speed to pellet them, remove the supernatant, and resuspend cells in fresh medium.
  Plate cells in culture vessels pre-coated with appropriate matrix proteins and incubate in a 37°C, 5% CO₂ atmosphere.