Technical library

Yes. RNAlater® Solution denatures proteins. 
As a result, proteins isolated from PromoCell cell pellets stored in RNAlater are suitable for applications, such as

• Western blotting
• 2‑D gel electrophoresis

and  not suitable for applications requiring native (functional) protein conformation, such as enzyme activity assays or protein–protein interaction studies.

PromoCell cell pellets (C-14**) are an easily accessible source of DNA, RNA, and proteins.

No, the PromoCell cell pellets (C-14**) are frozen at -20°C and cannot be revived. Their main application is to analyze RNA or protein. Cryopreserved cells that can be revived are available from the same donors. Please contact our Technical Customer Support if you need matched viable cells.

Slow growth after subculture can be caused by over-trypsinization or other suboptimal culture conditions. Please see attached trouble shooting guide for possible reasons.

Poor attachment after thawing can be a result of inappropriate freezing, storing or thawing the cells as well as from inadequate culture conditions (medium, incubator). The attached trouble shooting guide should help you to identify the possible reasons.

The difference between PromoCell DC Generation Medium Ready‑to‑use (C‑28050) and DC Base Medium (C‑28053) lies in the presence of cytokines and the degree of preparation:

• DC Base Medium (C‑28053) consists of 250 ml basal medium plus SupplementMix, but does not contain cytokines. It must therefore be supplemented with cytokines according to the user’s individual experimental needs.
• DC Generation Medium Ready‑to‑use (C‑28050) is a fully prepared medium and consists of 250 ml basal medium, SupplementMix, and the required cytokines. No additional supplementation is required before use.

PromoCell Monocyte Attachment Medium (C‑28051) is used when working with freshly isolated peripheral blood mononuclear cells (MNC) and a rapid, efficient adherence‑based selection of monocytes is required.
 
Purpose
The Monocyte Attachment Medium enables:

• Efficient adherence of monocytes within ~1 hour
• Selective enrichment of the monocyte fraction
• Preservation of optimal cell health

Key advantage
When using Monocyte Attachment Medium, time‑consuming and costly immunomagnetic monocyte purification steps are not required. The medium allows direct progression to downstream applications, such as macrophage or dendritic cell generation, without prior monocyte isolation.
 
Further information:
Detailed protocols for macrophage and dendritic cell generation from freshly isolated monocytes are provided in the corresponding PromoCell Application Notes.

• Generation of monocyte derived Dendritic Cells (moDCs)
• Differentiation of M1- or M2-Macrophages from PBMC/Monocytes

To differentiate the SkMC into myotubes, we recommend to use cells that have undergone a maximum of 4-5 population doublings. For this, the cells (ideally in P2 or P3) are cultured to 60-80% confluency in Skeletal Muscle Cell Growth Medium (C-23060). Then, a change to (serum-free) Differentiation Medium (C-23061) is performed to induce the differentiation process (formation of multinucleated syncytia). After 5 days, switch back to the Skeletal Muscle Cell Growth Medium for another 8 days to complete the differentiation. This protocol leads to stable myotubes and some of the myotubes show spontaneous contractions.

We have obtained best results when the cells have reached 60-80 % confluency. At this point, the Growth Medium is aspirated and replaced by Skeletal Muscle Differentiation Medium. After 2 - 8 days, extensive formation of multinucleated syncytia can be observed. For a stable differentiation of SkMC switch back to Skeletal Muscle Cell Growth Medium after 5 days incubation in Skeletal Muscle Differentiation Medium.

Yes, the Skeletal Muscle Cell Growth Medium can also be used for rat, mouse and rabbit SkMC. We recommend to use the medium right after isolation. Cells that were isolated and cultured in a different medium beforehand may have adapted to the other medium. An abrupt medium change causes stress to the cells resulting in reduced growth rates and lower differentiation capacities.