Aslamo alikom/Hello
I'm planning to do MTT experiment this week but unfortunately, we ran out of phenol-free media (PFM), which is added to MTT reagent for nearly 4 hrs. As it'll take awhile to order some, I'm wondering what might substitute PFM in the meantime.
Will PBS supplemented with FBS and GlutaMax work?
If not, what other temporary options available out there that will work just as well as PFM?
Thanks for your kind help in advance
Hello Mariam Ahmed Galal
Do you have HBSS (Hanks Balanced Salt Solution) without phenol red ?
HBSS is considered superior when it comes to preserving cell viability. It is non-toxic and has all nutrients, pH and osmolarity for cellular maintenance. The osmolarity of HBSS is found to be well within the range, enabling it to keep up the viability of cells for a period of at least two hours. HBSS will also maintain cellular integrity.
While PBS is a simple buffer, HBSS is more complex and includes components such as potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic and sodium bicarbonate. Hanks also contains glucose to help keep cells alive and happy for a longer time. If you keep the cells in PBS they can become stressed and die if left for too long.
You should give it a try with HBSS w/o phenol red but for not more than 2-3 hours. If required (not always necessary) you may supplement HBSS with a low concentration of FBS (0.2-0.5%) for cell survival.
Hope this will work!
Best.
Hi Mariam Ahmed Galal ,
When substituting phenol-free media for the MTT assay, you can use a variety of options, including:
1. RPMI 1640 without phenol red: RPMI 1640 is a commonly used medium for cell culture, and phenol-free versions are available. RPMI 1640 without phenol red can be used for the MTT assay without the interference of phenol red.
2. DMEM without phenol red: Dulbecco's Modified Eagle Medium (DMEM) is another widely used cell culture medium, and phenol-free formulations are available. DMEM without phenol red can be used as a substitute in the MTT assay.
3. Ham's F-12 without phenol red: Ham's F-12 medium is often used for a variety of cell types, and phenol-free formulations are available. Ham's F-12 without phenol red can be used as an alternative medium for the MTT assay.
4. Other custom or specialized phenol-free media: Depending on the specific requirements of your cell type or experimental conditions, there may be specialized phenol-free media available. These can include serum-free media, specific basal media for different cell types, or media designed for specific applications.
When selecting a phenol-free medium, ensure that it is compatible with your specific cell type and culture conditions. Also, check the composition and formulation of the medium to ensure it meets your experimental needs.
It is important to note that the MTT assay can be affected by various factors, such as the type of cells, cell density, incubation time, and MTT concentration. It is recommended to optimize the assay conditions when switching to a phenol-free medium to ensure consistent and reliable results.
Phenol-free media (PFM) is typically used in MTT assays to minimize interference with the formazan dye formation. While it is the recommended option, there are alternative solutions you can use temporarily if you don't have access to PFM. However, it's important to note that these alternatives may not provide the exact same level of performance as PFM.
Remember, while these alternatives can provide temporary solutions, it's important to confirm their compatibility with your specific cell lines and assay requirements. Once you are able to obtain phenol-free media, it is advisable to switch back to the recommended formulation for more accurate and reliable results in your MTT assay.
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