MOPS

MOPS is a buffering agent used in biology. MOPS buffer maintains the pH of mammalian cell culture media. MOPS significantly improves the thermal stability of BSA (HY-D0842)^[1][2][3]. In Vitro:Preparation of Keratinocyte and Microbial Culture Medium Using MOPS^[3]
Materials:
MOPS: 164 mM
RPMI-1640 medium
L-glutamine: 2 mM
Sodium bicarbonate: 2.0 g/L
Sterile phosphate-buffered saline (PBS, pH 7.2)
Normal oral keratinocytes (NOK) or human keratinocyte cell line (HaCat)
Microorganisms: Candida albicans SC5314, Staphylococcus aureus ATCC25923
Sterile water
Steps:
1. Preparation of MOPS Buffer:
Dissolve 164 mM of MOPS in 1 L of sterile water, and adjust the pH to 7.0.
Add 2 mM of L-glutamine and 2.0 g/L of sodium bicarbonate, stirring until fully dissolved.
Filter the solution using a sterile filtration device to ensure sterility.
2. Preparation of Keratinocyte Culture Medium:
Mix the prepared MOPS buffer with RPMI-1640 medium at a 1:1 ratio to obtain MOPS-containing medium.
Seed NOK or HaCat cells into a 24-well plate, adding approximately 4.5 × 10⁴ cells per well with an appropriate amount of RPMI/MOPS medium.
Incubate the cells in a 37°C, 5% CO₂ incubator until confluent (approximately 48 hours).
3. Preparation of Microbial Culture Medium:
Prepare the Candida albicans and Staphylococcus aureus strains, culturing them in suitable media until they reach the logarithmic growth phase.
Resuspend the microorganisms in MOPS medium at a concentration of approximately 10⁷ cells/mL.
Inoculate the microorganisms into a 24-well plate, adding an appropriate amount of MOPS medium, and incubate at 37°C in a shaker at 75 rpm for 90 minutes.
4. Co-culture of Keratinocytes and Microorganisms:
Co-culture keratinocytes and microorganisms in a 24-well plate, adding MOPS medium to maintain the pH around 7.0.
The co-culture duration can be set based on experimental needs, with a recommendation to limit the time to under 12 hours to reduce the cytotoxicity of MOPS.
5. Cell Viability Assay:
Use the MTT assay to measure cell viability, sampling every 2 hours, adding MTT/PBS solution, incubating at 37°C for 4 hours, and recording the results.
Notes:
MOPS is relatively safe for short-term experiments, but it may significantly reduce cell viability after 4 hours, so it is recommended to limit the experiment duration to 4-12 hours.