Technical library

Yes, you can aliquot the SupplementMix upon delivery and freeze down 2 or 4 individual aliquots at -20°C. This way, you can prepare smaller volumes (2 x 250 ml or 4 x 125 ml) of complete culture medium and thus extend the time you can use the medium.

At manufacture, ECGS (Endothelial Cell Growth Supplement) is adjusted to a protein content of 3 mg/ml.

• For Human Endothelial Cells and Microvascular Endothelial Cells, the optimal amount of ECGS is 2 ml ECGS per 500 ml medium (0.4% v/v), corresponding to 6 mg extracted protein per 500 ml medium.
• In the SupplementPack and Medium Kit, ECGS is supplied together with heparin (ECGS/H) in a single vial. The ECGS/H supplement contains 22.5 mg/ml heparin and provides a final heparin concentration of 45 mg per 500 ml medium (90 µg/ml) when added as recommended.

The Growth Medium Kit is recommended when flexibility in supplement concentrations is required.
It allows you to adjust the final concentrations of growth supplements, making it more flexible than the “ready‑to‑use” Medium. For example, it can be used to prepare customized media, such as a starvation medium.
Please note that modifying supplement concentrations may affect cell growth and viability. It is therefore recommended to test in advance whether and for how long the cells tolerate the altered culture conditions.

The three components of the PromoCell DetachKit (HEPES BSS, Trypsin/EDTA, and TNS) have a shelf life of 12 months from the date of manufacture when stored at -20°C.

After thawing
Once thawed for use, all components should be stored at 4-8°C and can be used for up to 6 weeks. Repeated freeze-thaw cycles should be avoided, as they may affect reagent performance.

Our Normal Human Cells have been cultured and tested in our growth media and have adapted to these conditions. Using other media may yield unsatisfactory results due to suboptimal supplies of nutrients and growth factors. PromoCell can only guarantee good cell growth (as stated in the Certificate of Analysis), when the cells are grown in the recommended media.

PromoCell Growth Media have special formulations and are much more complex than DMEM or RPMI + FBS. In comparison to immortalized cell lines, primary cells have much higher nutrient and growth factor requirements. Classical media do not usually achieve good performances with our cells.

PromoCell cell pellets are made from > 1 million cells and are dissolved in 200 µl RNAlater.

PromoCell Melanocyte Growth Medium M3 enables the serum‑, BPE‑, and PMA‑free cultivation of Normal Human Epidermal Melanocytes (NHEM) and does not require additional coating of standard tissue‑culture (TC) plastic.
 
Key considerations for optimal culture

• No coating required: NHEM can be cultured directly on standard TC‑treated plastic when using Melanocyte Growth Medium M3.
• Optimal confluency for passaging: To maintain the cells in a robust, adherent, and pro‑proliferative state, we strongly recommend passaging the cells at 70–90% confluency.
• Avoid high cell densities: High confluencies should be avoided, as elevated cell density can promote the formation of 3‑D spheroids. This phenomenon has been described in the literature for NHEM and may negatively affect monolayer culture and proliferation.

PromoCell strongly recommends using gentamicin at a final concentration of 50 µg/ml when isolating cancer stem cells (CSC) with the Primary Cancer Culture System.

Reason
Many commonly used antibiotics, such as penicillin or streptomycin, can inhibit the growth of primary cells, including primary cancer cells.
This inhibitory effect is particularly pronounced:
- at the clonal level
- at low cell density
Therefore, the use of antibiotics and antifungals should be approached with great caution in primary cancer cultures.

Recommendation
- Use gentamicin only (50 µg/ml) during isolation and culture.
- Avoid additional or alternative antibiotics whenever possible.

If antifungal protection is required
If the use of an antifungal agent is necessary, PromoCell recommends:
Caspofungin: 10 µg/ml (100× stock solution: 1 mg/ml in water)

Tumor‑associated macrophages (TAM) are an important component of the tumor microenvironment. Upon interaction with cancer cells, they typically adopt an anti‑inflammatory, pro‑tumor, and pro‑metastatic M2‑like phenotype.
For the isolation and culture of TAM from solid tumors, we recommend Primary Cancer Culture Medium supplemented with 250 ng/ml M-CSF, which supports TAM growth while reliably preventing stromal overgrowth.
The isolated TAM are non‑proliferating, adherent cells and can typically be cultured for at least two weeks.
 
Alternatively, the following media may be used:
- Macrophage Base Medium XF + 2% human AB serum
- M2‑Macrophage Generation Medium XF
However, please note that these media do not suppress fibroblast growth, resulting in a high risk of overgrowth by stromal cells.