Are your NHEK from the epidermis of the juvenile foreskin mucosal or cutaneous keratinocytes or a mixed population?

PromoCell's Normal Human Epidermal Keratinocytes (NHEK) from juvenile donors are isolated from both the epidermis of the outer and mucosal ( i.e. inner) layers of the foreskin. Thus, each vial contains a mixture of mucosal and cutaneous keratinocytes.

Do you recommend a certain negative control for the activated macrophages to compare the cytokine release with?

Even non-activated macrophages do release a certain amount of cytokines. Furthermore, you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore, we do not think it is possible to have a general negative control for the cytokine release.

Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?

No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.

Which cytokine concentrations does PromoCell recommend for activating M1/M2 macrophages?

You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.

Did PromoCell try plating the cryo-macrophages in 96-well plates?

We did not plate the cryo-macrophages in 96-well plates. However, we heard from other customers that they have successfully used our macrophages in this kind of plate.

Are endothelial cells α-SMA-negative under all circumstances?

Scientific findings from different groups, as well as our own results indicate that the presence or absence of α-SMA is not a valid indicator for the composition of a given cell population. Research findings and our inhouse data suggest that endothelial cells are not α-SMA-negative under all circumstances. α-SMA negativity is not an intrinsic property of endothelial cells but can vary depending on extrinsic influences. Lot-specific values for α-SMA in our Certificates of Analysis (CoAs) are therefore not considered meaningful. Further Information Ando et al.; Am J Physio l. 1999 May;276(5):H1755-68 Lu et al.; Stem Cells Dev . 2004 Oct;13(5):521-7 Azuma et al.; Biochem Biophys Res Commun . 2009 Mar 13;380(3):620-6 Frid et al.; Circ Res . 2002 Jun 14;90(11):1189-96 Kovacic et al.; J Am Coll Cardiol . 2019 Jan 22;73(2):190-209 Jones et al.; Am J Respir Cell Mol Biol . 1999 Apr;20(4):582-94 Ma et al.; Front Cell Dev Biol . 2020 Apr 21;8:260 Kocher and Madri; In Vitro Cell Dev Biol . 1989 May;25(5):424-34 Amberger et al.; FEBS Lett . 1991 Aug 5;287(1-2):223-5

Is vWF a great marker for Endothelial Cells?

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (vWF) are more linked to the formation of a confluent endothelial monolayer. Research shows that vWF expression is dynamic and highly dependent on the cell culture conditions such as confluence and passage number. Therefore, having vWF as a quality control marker for each lot of ECs is not really necessary. Further Information Howell et al.; Mol Membr Biol. Nov-Dec 2004;21(6):413-21

Can I defrost Accutase Solution, prepare aliquots and refreeze them?

Yes, Accutase Solution can be defrosted, aliquoted, and then refrozen. Defrosting: Accutase should be defrosted overnight in the refrigerator or placed in a tub of cold tap water. Do not defrost in a 37°C water bath. Stability: Once thawed, it is stable for at least 2 months in the refrigerator if stored promptly after use.

Is it possible to refreeze the hCD34 progenitor cells after having amplifed them in PromoCell Hemaotopoietic Progenitor Cell Expansion Medium XF?

Yes, it is possible to refreeze them.

If I isolate fresh CD14-monocytes for M1/M2 macrophage generation, can I culture them for a period of time before differentiation?

This is not advised. Please seed the freshly isolated CD14-monocytes immediately in the Monocyte Attachment Medium. Adding a culturing step will change the biological characteristics of monocytes very rapidly.

Why is PBS added in the last step of the Alizarin Red staining protocol (Osteogenic differentiation and analysis of MSC)? Should the PBS be aspirated before analysis?

The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense. Leave the PBS on the cells after staining/washing and analyze the sample immediately, as the dye may bleed upon prolonged storage without embedding.

Technical library - RESOURCES

The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time, increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop. Mycoplasma infections can also occur more easily, as they are often introduced along with contaminants such as bacteria and fungi. Last but not least, antibiotics are known to have negative effects on the metabolism of eukaryotic cells - more details on this topic can be found in our blog article "Antibiotics in cell culture: friend or enemy".

Further Information

Blog article: Antibiotics in Cell Culture: Friend or Enemy?