Technical library

PromoCell uses a classification system similar to, but not identical with, the Fitzpatrick Skin Classification.

The Fitzpatrick classification distinguishes six phototypes (I–VI) based on skin pigmentation (melanin content) and the skin’s response to sun exposure:

• Phototype I: Very fair skin, always burns, never tans
• Phototype II: Fair skin, burns easily, tans poorly
• Phototype III: Medium skin, tans after initial burn
• Phototype IV: Light brown skin, rarely burns, tans easily
• Phototype V: Brown skin, very rarely burns, tans darkly
• Phototype VI: Dark brown or black skin, never burns

PromoCell donor classification:
For PromoCell skin tissue donors, information is available on:

• Skin color (white, brown, or black)
• Eye color
• Hair color

However, no information is available on individual tanning or burning behavior.
Therefore, PromoCell classifies donors into the following three phototype groups:

• Light (corresponding to Fitzpatrick phototypes I and II)
• Moderate (corresponding to Fitzpatrick phototypes III and IV)
• Dark (corresponding to Fitzpatrick phototypes V and VI)

Information on the phototype group is available for most cell lots derived from juvenile or adult skin tissue. Our Scientific Support team can assist in the selection of suitable cell lots based on the desired donor phototype.

a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven't tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%). Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g., evaporation of media, dry wells, etc.).

You should use complete DC Generation Medium/DC Generation Medium XF (with all the cytokines). As cells are metabolically active, media should be changed every 3 days. We have observed that the dendritic cell phenotype remains stable for up to 7 days.

The Macrophage Detachment Solution (C‑41330) acts directly on the cell membrane to enable efficient detachment of macrophages.
Following detachment, the cells are in a particularly sensitive state. The addition of Human Serum Albumin (HSA) to the PBS wash buffer serves an important protective function:

• HSA supports regeneration and stabilization of the cell membrane
• It protects macrophages from detrimental effects during the critical phase immediately after detachment

This helps to maintain cell integrity and viability prior to downstream applications.

Our HDMEC pre-screened cells are isolated from juvenile foreskin.

PromoCell Human Pulmonary Microvascular Endothelial Cells (HPMEC, C‑12281) are isolated from the lung parenchyma.
During the isolation process, all large vessels are removed beforehand. As a result, the majority of HPMEC originate from pulmonary capillaries.

There are two options for isolating RNA from cells stored in RNAlater Solution: 1) The solution is removed from the cells prior to extraction by centrifugation at 5,000 x g for 10 minutes at 4°C. Note: Because of the density of RNAlater® solution, greater centrifugal forces are required to spin down the cells. 2) If no pellet is visible after centrifugation, RNA can also be purified directly from the RNAlater® solution. This can be done by adding 2 ml of 10x lysis buffer, and proceeding normally.

For chondrogenic differentiation of human mesenchymal stem cells (hMSC), it is essential that the cells do not adhere to the culture surface.
During differentiation, hMSCs aggregate and form spheroids that float freely in the medium. Therefore:

• No specific plate brand is required
• Any 96‑well U‑bottom plate that is suitable for suspension culture can be used
• The critical requirement is the U‑bottom geometry, which supports spheroid formation and prevents cell attachment

We have tested the differentiation capacity of our hMSC into adipocytes, chondrocytes and osteoblasts over time and still see good differentiation rates after 10 population doublings, i.e., at passage 5. However, the differentiation potential declines with ongoing population doublings. To obtain optimal differentiation rates, experiments should be performed as early in culture as possible.

• Adipogenic differentiation can be identified morphologically and without any staining by the formation of intracellular lipid vesicles.
• In contrast, when MSC differentiate into bone cells, there is no significant change in morphology. It is recommended to perform Alkaline Phosphatase staining to detect osteoblastic differentiation or Alizarin Red S staining to show osteoblast mineralization.
• Chondrogenic differentiation is generally performed as spheroids in 3-D cell culture and not in 2-D monolayer culture. Staining with Alcian Blue to visualize the differentiation process is indispensable.
• Neurogenic differentiation can be detected using neuron specific markers (e.g. beta-3 tubulin, NeuN, MAP2) and by their typical neuronal morphology.

For differentiation protocols, please see attached Application Notes.