Technical library

If hematopoietic progenitor cells are cultured in Hematopoietic Progenitor Cell Expansion Medium XF (C‑28021) without Cytokine Mix E (C‑39890), the cells will not survive and will die.
Hematopoietic Progenitor Cell Expansion Medium XF must always be supplemented with Cytokine Mix E or, alternatively, with an appropriate, functionally equivalent cytokine cocktail to support cell survival and proliferation.

The difference between PromoCell DC Generation Medium XF (C‑28052) and DC Base Medium XF (C‑28054) is the presence of cytokines and the level of preparation:

• DC Base Medium XF (C‑28054) consists of 250 ml basal medium plus SupplementMix, but does not contain cytokines. It must therefore be supplemented with cytokines according to the user’s individual experimental requirements.
• DC Generation Medium XF (C‑28052) is ready‑to‑use and consists of 250 ml basal medium, SupplementMix, and the appropriate cytokines required for dendritic cell generation. No additional supplementation is needed before use.

Both DC Base Medium XF and DC Generation Medium XF feature a xeno‑free formulation.

PromoCell DC Generation Medium XF (C‑28052) is specifically designed for the in vitro generation of monocyte‑derived dendritic cells (moDCs) from freshly isolated peripheral blood monocytes. The key advantages are:
 
Xeno‑free formulation
The medium is xeno‑free, eliminating animal‑derived components and reducing variability associated with serum‑ or animal‑based supplements. This supports higher reproducibility and compatibility with defined culture conditions.
Complete, ready‑to‑use system
DC Generation Medium XF is supplied as a ready‑to‑use medium with all required cytokines included, simplifying handling and reducing preparation steps and potential sources of error. 
Efficient and reproducible moDC generation
The medium enables efficient and reproducible differentiation and maturation of monocyte‑derived dendritic cells (moDCs) from freshly isolated peripheral blood monocytes, supporting consistent experimental outcomes.

Yes. PromoCell Lymphocyte Separation Medium 1077 (C‑44010) is endotoxin‑tested.
The product specification is < 0.3 EU/ml.

Airway organoids such as bronchospheres can be cultured in 96‑well U‑bottom plates designed for suspension cells.

• A commonly used example is 96‑well U‑bottom suspension plates (e.g. Greiner Bio‑One #650185).
• A detailed protocol for culturing airway organoids in 96‑well plates is provided in the corresponding PromoCell Application Note "Generation of human airway organoids from primary cells". 

Plate formats not tested
384‑well plates and other commercially available plate formats have not been validated by PromoCell for this application.

ROCK inhibitors (ROCKi), such as Y‑27632, are widely used to enhance epithelial cell and keratinocyte progenitor proliferation and to improve cell survival through a process known as conditional reprogramming. Importantly, this effect is reversible.
In addition, ROCKi is known to:

• Increase seeding efficiency on plastic surfaces
• Support early survival of epithelial cells during stressful steps (e.g. dissociation, seeding)

ROCKi in airway epithelial cells and organoids
The effects of ROCKi‑mediated conditional reprogramming have also been described for airway epithelial cells (EpCs) and airway organoid cultures, as reported in the literature:

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

PromoCell recommendation
For human airway organoid cultures, the use of Y‑27632 ROCKi is optional, as organoids are able to form even without ROCKi. However, 3‑D culture systems can be sensitive to factors such as:

• the choice of plastic
• the extracellular matrix (ECM) used

Under these conditions, the addition of ROCKi may help to increase robustness and reproducibility of the system, particularly during the initial phases of organoid establishment.

Possibly. Airway organoid size and size distribution may be influenced by nutrient availability, particularly within the extracellular matrix (ECM) gel.
A published study suggests that organoid size can be affected by nutrient access in the matrix.
 
Practical recommendations
Based on our experience, the following points can help improve size consistency:
1. Avoid nutrient gradients: 

• Use small, drop‑like BME gel beads
• Combine with relatively high medium volumes
• Change the medium daily, as less frequent changes can result in yellow medium, indicating a pH shift likely caused by increased cellular metabolism

2. Control ECM gelling carefully:
Temperature is critical:

• BME is liquid at 4°C
• BME solidifies at 37°C

Gelling must occur rapidly; otherwise, cells may sink to the bottom of the gel, leading to uneven cell distribution and variable organoid sizes.

Yes. Airway organoid orientation can be influenced by the use or absence of an extracellular matrix (ECM).

• Without ECM, organoids tend to show a higher proportion of outward‑oriented ("apical-out") polarity.
• A published approach describes generating organoids initially embedded in ECM, followed by ECM dissociation and re‑seeding of free organoids in suspension without ECM, which promotes outward orientation. The protocol is described here.

Please note: This method has not been tested internally at PromoCell, and therefore cannot be guaranteed.

For quality control of PromoCell keratinocytes, we use a pan‑cytokeratin antibody rather than antibodies against individual cytokeratin subtypes.

• The pan‑cytokeratin antibody detects a broad range of cytokeratins
• This approach reliably confirms the epithelial identity of keratinocyte cultures

PromoCell guarantees more than 500,000 viable cells per vial.
To reliably meet this guarantee, more than 500,000 cells must be frozen at the time of cryopreservation. This is necessary because the post‑thaw viability cannot be determined at the moment of freezing.

Additional factors
For technical and organizational reasons, the initial cell number may vary between different lots. As a result:

• Two lots with the same post‑thaw viability may still contain different absolute cell numbers.
• The actual number of cells in a vial may therefore be significantly higher than 500,000.