Technical library - RESOURCES

The Monocyte Attachment Medium allows for the efficient adherence selection (within 1 hr) of monocytes from freshly isolated mononuclear cells while maintaining optimal cell health. The Time-consuming and costly immunomagnetic purification of monocytes prior to DC generation is not necessary and can be skipped when using this medium.

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.

The following criteria are applied for blood donations: a) Donors with light infection like a cold or cough are excluded for one week b) Donors with infections with a temperature above 37.9°C and/ or antibiotics therapy are excluded for 4 weeks c) Blood pressure: exclusion only if systolic blood pressure is < 100 mm Hg or > 180 mm Hg or if diastolic blood pressure is > 100 mm Hg. No exclusion if blood pressure is drug treated and in acceptable range. d) High cholesterol: no exclusion even though medication is used e) Diabetes: exclusion if diabetes type I or use of insulin f) Steroid use: exclusion for 4 weeks after application g) Cancer: exclusion h) Other chronic diseases: exclusion depends on the type of disease (for example chronic heart disease, autoimmune disease)

Our SkMC are proliferating myoblasts that have retained the capacity to differentiate. Upon withdrawal of serum and growth factors, differentiation is induced and the cells form multinucleated syncytia.

For efficient differentiation of our SkMC into myotubes, we recommend to use cells that have undergone a maximum of 4-5 population doublings, i.e. not more than 1 additional subculturing step after thawing the original vial. For more details about differentiation, please see the instruction manual of our Skeletal Muscle Cell Media.

At PromoCell, we get the umbilical cords from our tissue suppliers with no addition of buffers or media. This method prevents the microorganisms from being washed into the blood vessels. Before we start the cell preparation, the umbilical cord is also cut on both ends with a sterile scalpel to provide sterile intersections in addition to the sterile lumen. This method allows us to isolate sterile endothelial cells from umbilical vein and to plate them in antibiotics-free culture media.

The PromoCell quality control procedure includes the cultivation of fibroblasts for 15 population doublings. This is to ensure that the cells can be grown for a minimum of 15 PDs (6-8 passages depending on the split ratio used) but it does not mean that the cells immediately senesce after that point. We don't determine the maximum number of doublings or passages but most NHDF lots will certainly achieve > 20 passages.

Normal osteoblasts, similar to other non-transformed cell types can be expanded in vitro to a certain extent before they are used for experiments. Nonetheless, HOB are generally used at low passages (up to P4) in most labs.

Short protocol:

Wash the cells with sterile PBS (w/o Ca++/Mg++) or HepesBSS
Add undiluted accutase to the culture vessel (2 ml per 25 cm²)
Incubate at room temperature for 5-15 min or at 37°C for faster detachment
When the majority of the cells has detached, centrifuge the suspension and resuspend the pellet in fresh medium. In most cases, no additional washes or neutralization steps are required.

Soluble receptors are post-translationally modified and generally contain disulfide bonds and glycosylation sites. If produced in E. coli the proteins don't show any biological activity.