Technical Library

Recent Entries in Technical Library

• Why use ROCKi for organoid formation?

  ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic.

  This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:

  • https://pubmed.ncbi.nlm.nih.gov/31871466/
  • https://pubmed.ncbi.nlm.nih.gov/35169685/

  For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• Can you control size / size distribution of organoids?

  Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/

  For best results we recommend the following:

  • Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
  • For optimal cell distribution,  it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• Can you drive organoid orientation (inward vs outward)?

  Yes. The orientation can be triggered by the use or lack thereof of ECM.

  Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/, where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM.

  * Please note that we have not tested this method in our labs and thus cannot guarantee it.

  Related Links and Documents

  • AppNote: Generation of Human Airway Organoids from Primary Cells
• For which cytokeratin are the keratinocytes tested?

  PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.

  Related Links and Documents

  • Normal Human Epidermal Keratinocytes
  • Normal Human Epidermal Keratinocytes GM3
• Why is the actual cell count in the vial at PromoCell sometimes significantly higher than the 500,000 cells stated on the website?

  We guarantee > 500,000 viable cells per vial. To do this, we need to freeze more than 500,000 cells, as we do not yet know the viability after thawing at the time of freezing.
  For technical/organizational reasons, the initial cell number may also vary from lot to lot, so 2 lots with the same viability may not necessarily contain the same number of cells.

  Related Links and Documents

  • Human primary cell culture
• Can your Macrophage Detachment Solution also be used for bone marrow-derived macrophages from mice?

  Yes, there is a reference (Campuzano et al.; J Immunol. 2020 Jun 15;204(12):3296) where bone marrow-derived macrophages from mouse were detached using our Macrophage Detachment Solution (40 minutes at 4°C).

   

  Related Links and Documents

  • Macrophage Detachment Solution
  • Campuzano et al.; J Immunol . 2020 Jun 15;204(12):3296
• 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.

   

   

  Related Links and Documents

  • NHEK
  • NHEK in GM3
• 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.

   

  Related Links and Documents

  • Application Note: Osteogenic Differentiation and Analysis of MSC
• What are the disadvantages of the prophylactic use of antibiotics in cell culture?

  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”.

  Related Links and Documents

  • Blog article: Antibiotics in Cell Culture: Friend or Enemy?
• I have accidently stored the Cryo-SFM at -20°C. Can the product still be used in this case?

  According to the product manual, Cryo-SFM should be stored at 4-8°C. However, since this solution is used to freeze cells in liquid nitrogen, we assume that storing Cryo-SFM once at -20°C should not have a negative impact on the product quality. After thawing, please store it at 4-8°C as recommended.

  Related Links and Documents

  • Freezing Medium Cryo-SFM