Where in the lung are the HPMEC harvested from? Are the cells from arterioles or capillaries?

Our human pulmonary microvascular endothelial cells (C-12281) are sourced from the lung parenchyma with all large vessels being removed beforehand. Therefore, most of the HPMEC originate from capillaries.

How do I best isolate RNA from PromoCell cell pellets (C-14**)?

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.

Which plates does PromoCell recommend as 96-well U bottom suspension plates for chondrogenic differentiation of Human Mesenchymal Stem Cells?

For chrondrogenic differentiation it is important that the cells do not adhere to the wells. During differentiation, the cells form spheroids which float in the medium. Therefore, there are no special requirements for the wells as long as they are U bottom shaped and suitable for suspension culture.

Does the differentiation capacity of Human Mesenchymal Stem Cells change over time? When should I best induce differentiation?

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.

Can I detect the differentiation of Mesenchymal Stem Cells by their morphology, without doing any cell staining?

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.

How do mesenchymal stem cells from different tissues differ in terms of their biological function?

The human MSC derived from bone marrow, adipose tissue, and umbilical cord matrix are from different origins, but with comparable biological properties and function. Depending on the tissue of origin, they may have a higher preference for differentiation into one particular cell type and a lower preference for another one, but they all still retain the differentiation potential for the mesenchymal lineage. MSC-BM: very good differentiation into bone cells, chondrocytes & fat cells MSC-UC: very good differentiation into chondrocytes; but weaker potential into fat/bone cells MSC-AT: very good differentiation into fat cells

Can I expand the human Mesenchymal Stem Cells prior to differentiating them?

These cells are frozen at the end of 2 nd culture. Thawing and seeding results in passage 2 (3 rd culture). We recommend that they be used for differentiation experiments not later than passage 5. The differentiation potential of hMSC in vitro is reduced with ongoing population doublings, meaning the earlier differentiation is induced, the higher the differentiation rates.

To what extent will the CD34+ Progenitor Cells proliferate in Hematopoietic Progenitor Cell Expansion Medium XF (C-28021)?

For optimal cell growth, the human CD34+ Progenitors (C-12921) are cultured in PromoCell Hematopoietic Progenitor Cell Expansion Medium XF (C-28021) supplemented with Cytokine Mix E (C-39890) or with any other cytokine cocktail suitable to achieve optimal cell expansion. Cytokine Mix E contains recombinant human TPO, SCF, flt3-ligand and IL-3. The strong expansion of CD34+ cells will persist for approx. 2 weeks. The expansion factor for CD34+ cells is usually between 75 and 200-fold.

I would like to know the number of population doublings for PromoCell mononuclear cells (C-12907/C-12901) when grown in Mononuclear Cell Medium (C-28030).

Mononuclear cells are mostly used in immunology, infection biology, hematology and cancer research to study subpopulations of blood cells. Our Mononuclear Cell Medium (C-28030) is intended for short-term maintenance (up to 48 hrs) of the thawed hMNC before you proceed with your experiments. The number of PDs will depend on the subsequent cell culture conditions and is not determined by PromoCell. Please note: Depending on the conditions, the ratio of the subpopulations will gradually change, as the different blood cell types behave in different manners. Researchers normally start soon after thawing to either select the cell type of their interest (e.g. hematopoietic cells, endothelial progenitor cells) or perform experiments with all populations of hMNCs (e.g. to study effects on toxicity, viability or metabolism).

What plating density should I use for PromoCell Human Adult Stem and/or Blood Cells?

The recommended plating density after thawing/subculture may vary depending on the cell type. It is specified on the Website (tab: additional Information) and in the Product Manual.

I have a question related to PromoCell's CD34+ Progenitor Cells (C-12921). Are they suspension cells or do they partially attach?

It actually depends on the culture conditions whether the cells remain in suspension or attach to the surface. When grown in our serum-free, xeno-free HPC Expansion Medium XF (C-28021), the cells remain in suspension.

What plating density should I use for PromoCell Normal Human Cells?

The recommended plating density after thawing/subculture may vary depending on the cell type. Please refer to the data sheet for your cells under "Specifications".

Technical library - RESOURCES

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.