Technical library

Yes, PromoCell Normal Human Cells can be re-frozen after thawing and expansion. However, re-freezing is generally not recommended because each additional freeze-thaw cycle can reduce cell recovery, viability, and proliferation potential.
Please note that after an additional freezing cycle, the number of population doublings (PDs) specified in the Certificate of Analysis (CoA) can no longer be guaranteed.
If re-freezing is required, we recommend freezing the cells at a low passage number and using PromoCell Cryo-SFM Plus (C-29920). Cryo-SFM Plus is a defined, animal component-free, and protein-free cryopreservation medium developed to support optimal storage and recovery of primary human cells.
For best results, avoid unnecessary freeze-thaw cycles and establish working stocks from early-passage cultures whenever possible.

No. At day 3 of the dendritic cell (DC) generation protocol, there should be no significant phenotypic difference between the non-adherent cells and the loosely attached dendritic cells.
If you would like to assess the phenotype at this stage, gently rinse the culture surface to detach a portion of the loosely attached cells and combine them with a representative fraction of the non-adherent cells for downstream analysis. This approach provides a sample that reflects the overall dendritic cell population present in the culture at day 3.
Note: Loosely attached cells at this stage are typically considered part of the developing dendritic cell population and can be analyzed together with the non-adherent fraction.
Further reading: For comprehensive guidance on monocyte-derived dendritic cell (moDC) generation, maturation, morphology, and phenotypic characterization, please see our Application Note Generation of Monocyte-Derived Dendritic Cells (moDCs).

For PBMC isolation using 3 ml of Lymphocyte Separation Medium 1077 (C-44010) in a 15 ml conical tube, we recommend loading 3-8 ml of diluted whole blood onto the separation medium.
Before layering, the whole blood should be diluted with PBS at a ratio of 1:2 or 1:3.
 
Example (1:2 dilution):

• Mix 1 volume of whole blood with 1 volume of PBS.
• To obtain 3-8 ml of diluted blood, use 1.5-4 ml of whole blood and add an equal volume of PBS.

As a general guideline:

• 15 ml conical tube: 3 ml Lymphocyte Separation Medium 1077 + 3-8 ml diluted whole blood.
• 50 ml conical tube: 15 ml Lymphocyte Separation Medium 1077 + 15-30 ml diluted whole blood.

For optimal separation, carefully layer the diluted blood onto the Lymphocyte Separation Medium without disturbing the interface.

PromoCell Human Pulmonary Artery Endothelial Cells (HPAEC, C-12241) are isolated from the pulmonary arteries of adult donors. The source tissue is obtained through forensic medicine. Therefore, HPAEC are considered adult primary endothelial cells and are not of fetal or neonatal origin.
Adult HPAECs provide a physiologically relevant model for studying pulmonary vascular biology, including endothelial barrier function, inflammation, and vascular remodeling. They are mainly used in research to study Pulmonary Arterial Hypertension (PAH), Pulmonary Fibrosis, Acute Lung Injury (ALI), and Acute Respiratory Distress Syndrome (ARDS).

Yes. PromoCell Melanocyte Growth Medium M3 (C-24310) can be used for the isolation of primary human melanocytes.
At PromoCell, Melanocyte Growth Medium M3 is routinely used for the isolation and cultivation of Normal Human Epidermal Melanocytes M3 (NHEM M3) from human epidermis. The medium supports the selective growth of melanocytes and is suitable for establishing primary melanocyte cultures following tissue isolation.
For optimal results, please follow the recommended cultivation protocol and use appropriate aseptic cell culture techniques throughout the procedure.

Passage number and population doublings (PDs) describe different aspects of cell culture expansion.

• Passage number indicates how many times cells have been subcultured (detached and re-seeded).
• Population doublings (PDs) indicate how many times the cell population has doubled in number.

Unlike passage number, PDs reflect the actual proliferation and biological age of a cell culture. For example, a 1:2 split and a 1:6 split are both counted as one passage, but they result in different levels of cell expansion and therefore different numbers of PDs.
For this reason, PromoCell specifies and guarantees population doublings (PDs) rather than passage numbers. Under typical culture conditions, 15 PDs correspond to approximately 6-8 passages, depending on the split ratio, seeding density, and cell yield.
 
Summary
Passage number counts subculturing events, whereas PDs measure actual cell proliferation. Therefore, PDs provide a more meaningful indication of culture age and expansion history.

Seeding density refers to the number of cells plated per unit surface area (typically cells/cm²) and is an important factor for cell attachment, growth, viability, and reproducibility. The recommended seeding density provided in the Instruction Manual has been optimized for the respective primary human cell type and generally applies to both the initial seeding of thawed cells and routine subculture.
After thawing, cell recovery and attachment rates may vary, and some cell loss is expected due to the stress associated with cryopreservation and thawing. Using the recommended seeding density helps ensure that sufficient viable cells attach, recover, and establish a healthy proliferating culture.
During routine subculture, the same seeding density supports consistent growth and helps maintain the culture conditions validated for the respective cell type. Following the recommended density can reduce experimental variability and promote reproducible results. A slightly lower seeding density may also be appropriate, provided that healthy and consistent cell growth is maintained.
Our internal experience shows that many cell types can be successfully cultured at seeding densities of approximately 5,000 cells/cm². Experienced users often achieve good results with similar densities after thawing and during subculture. However, lower seeding densities may be associated with slower culture establishment and extended recovery periods.
For optimal performance, always follow the latest Instruction Manual supplied with your cells and culture medium. If you are unsure which seeding density is most appropriate for your application, the PromoCell Scientific Support team will be happy to provide cell type-specific recommendations and culture optimization guidance.