Technical library

Please follow the instructions below when handling PromoCell proliferating cells upon arrival.

Initial handling after delivery
1. Unpack the shipment immediately and place the T25 flask(s) into a 37°C incubator with the cap closed.
2. Allow the cells to recover for approximately 3 hours.
3. After incubation, check the cell confluency under the microscope.

Medium change
If the cell density is below 70% confluency, aspirate the medium under sterile conditions and add 5-10 ml of the appropriate Growth Medium.

Subculturing
Once the cells have reached more than 70% confluency, they should be subcultured according to the subcultivation protocol provided in the Instruction Manual of the respective cell type.
 

Under standard culture conditions, PromoCell Normal Human Chondrocytes (HCH, C‑12710) typically reach subconfluency within ~4-7 days after seeding.
The number of population doublings (PDs) per passage depends on the seeding density and the cell yield at subconfluency and can be calculated accordingly.
As a general guideline:
When HCH are seeded at 10,000 cells/cm², they typically undergo approximately 2 population doublings per passage.

PromoCell Human White Preadipocytes (HWP) are isolated from defined adipose tissue depots, depending on the product variant:
 
Subcutaneous HWP (C-12735)
Isolated from subcutaneous fat of different anatomical localizations, such as the abdomen, breast, or upper arm.
 
Visceral HWP (C-12732)
Isolated from visceral fat, for example: fat surrounding the pericardium, or adipose tissue from the omentum or mediastinum.
 
The exact tissue localization for each cell lot is specified in the lot‑specific Certificate of Analysis (CoA). If you require HWP from a specific anatomical localization, please contact the PromoCell Scientific Support Team before placing your order, as availability depends on donor material.

The time required to detach primary cells by trypsinization varies and depends on multiple factors, including cell type, cell density, cell lot, trypsin concentration, efficiency of the washing step before adding trypsin, and trypsinization temperature.
Because of this variability, a single fixed trypsinization time would not be reliable or safe for all situations.
 
PromoCell recommendation
For most primary cell types, PromoCell recommends:

• Trypsinization at room temperature
• Continuous microscopic monitoring of cell detachment

This approach allows you to determine the optimal trypsinization time for your specific culture and to keep the contact time between cells and trypsin as short as possible, minimizing cell damage. In practice, most cells detach within approximately 2-8 minutes.
 
Cell‑type‑specific exceptions
For certain cell types:

• Trypsinization at 37°C, or
• the use of Accutase or another dedicated Detachment Solution

may be recommended. Please refer to the Instruction Manual of the respective cell type for detailed guidance.
 
 

The choice of multiwell plate depends on the detection method, as plate color and opacity strongly influence signal quality and background noise.
1) Fluorescence detection (fluorometer)
- Recommended plates: Black plates with clear bottoms
- Reason: Black walls reduce well‑to‑well crosstalk and background fluorescence, while clear bottoms allow bottom‑read detection.
- Note: In some applications, clear plates may also be sufficient, depending on signal strength and assay sensitivity.
2) Luminescence detection (luminometer)
- Recommended plates: White or opaque plates
- Reason: White plates reflect emitted light and maximize signal intensity, improving sensitivity for low‑light luminescence assays.
3) Colorimetric detection (photometer/absorbance reader)
- Recommended plates: Clear plates
- Reason: Clear wells are required for accurate absorbance measurements, as light must pass directly through the sample.

Both NHEK.f and NHEK.f pooled are Normal Human Epidermal Keratinocytes derived from juvenile foreskin, but they differ in their donor composition and preparation.

NHEK.f single donor (C-12001)

• Isolated from the foreskin of a single donor
• Donor age range: 1 -  ~10 years
• Cells reflect the biological characteristics of one individual donor
• After thawing, cells are at passage 2 (P2)

NHEK.f pooled (C-12005)

• Prepared from the foreskins of two or three individual donors
• Cells from each donor are expanded separately in individual tissue‑culture vessels, and pooled after secondary culture, prior to cryopreservation
• Pooling reduces donor‑to‑donor variability
• After thawing, cells are also at passage 2 (P2)

Summary

• NHEK.f: single‑donor cells → suitable for donor‑specific studies
• NHEK.f pooled: multi‑donor pool → suitable for more standardized and robust experiments

After isolation and purification, PromoCell primary human melanocytes are characterized during quality control to confirm their identity.
 
How melanocyte identity is confirmed

• Cells are checked for their typical melanocyte morphology
• Expression of the marker Mel‑5 is verified: Mel‑5 is a 75 kDa glycoprotein that is typically expressed by normal human melanocytes

These criteria ensure the identity and purity of the supplied melanocytes.
 
Selectivity of melanocyte media
 
1. Melanocyte Growth Medium (C‑24010)

• Developed to promote melanocyte growth in vitro
• However, it does not completely inhibit the growth of other cell types, such as normal human epidermal keratinocytes (NHEK) or normal human dermal fibroblasts (NHDF).
• Therefore, it is essential to start with a pure melanocyte culture when using this medium. 

2. Melanocyte Growth Medium M3 (C‑24310)

• Much more selective for melanocytes
• More effectively suppresses the growth of contaminating cell types
• Provides improved robustness when working with primary melanocyte cultures

Yes. PromoCell Skeletal Muscle Cell Differentiation Medium (C‑23061) is completely defined.
The included SupplementMix (C‑39366) contains recombinant human insulin as its functional component.

No. Heat inactivation of fetal calf serum (FCS/FBS) is generally not necessary when working with Normal Human Cells.
 
Background
The practice of heat inactivation was originally introduced when serum from adult animals was commonly used. Adult serum can contain high levels of complement, which may damage cells under certain conditions. Heating serum (typically 30 min at 56°C) was intended to inactivate complement activity.
 
Current practice
FBS contains very low complement activity compared to adult serum.
Today, FBS is often heat‑inactivated without clear evidence of a beneficial effect.
For most cell types and cell lines, including Normal Human Cells, heat inactivation of FCS is not required.
 
PromoCell practice
PromoCell does not use heat‑inactivated serum for the production of its growth media.

No. After adding the SupplementMix or SupplementPack to the corresponding PromoCell basal medium, you obtain a complete culture medium.
No additional supplementation with fetal calf serum, growth factors, or L-glutamine is required, as all components are already present at optimized concentrations.
 
Important note on antibiotics

• PromoCell media do not contain antibiotics.
• If desired, penicillin/streptomycin or gentamicin/amphotericin B can be added at standard concentrations.
• Please note that the use of antibiotics may reduce cell doubling time by up to 20%.