Technical library

Most primary cells detach within approximately 5–15 minutes at 37°C when using accutase solution (C‑41310).
 
Additional notes

• Inactivation of accutase is not required
• Once most of the cells have detached, collect the cell suspension, dilute it with fresh medium, and centrifuge it to remove accutase and EDTA before replating the cells

This helps ensure optimal cell recovery and performance in subsequent culture steps.

PromoCell Human White Preadipocytes (HWP) are isolated from either subcutaneous or visceral fat tissue, which differ in location, physiological role, and disease relevance.

Subcutaneous fat
Location: Directly beneath the skin
Physiological role: Acts as the primary and physiological energy storage depot
Disease association: Generally not directly linked to obesity‑related metabolic diseases
Use case: Suitable for studies on normal adipocyte biology, energy storage, and baseline adipogenesis

Visceral fat
Location: In the abdominal cavity, surrounding internal organs (e.g. kidney, heart, bladder)
Physiological role: Expands when subcutaneous storage capacity is exceeded or adipogenesis is impaired
Disease association: Strongly linked to hypertension, type 2 diabetes, and cardiovascular disease
Use case: Preferred for research on metabolic disorders, insulin resistance, inflammation, and cardiometabolic risk
 
Functional differences
Adipocytes derived from subcutaneous and visceral fat differ in several functional aspects, including:

• Response to insulin and other hormones
• Lipolytic activity
• Endocrine and metabolic behavior

The choice between the two cell types therefore depends on the study objective.

Number of cells per cryovial
PromoCell guarantees ≥ 500,000 viable primary human cells after thawing. To ensure this, more than 500,000 cells are filled per cryovial before cryopreservation, since a fraction of cells does not survive the freeze-thaw process.
 
Viable cell number and Certificate of Analysis (CoA)
During quality control, PromoCell thaws representative vials per lot, determines viability, and calculates the number of viable cells recovered after thawing.
Both the viable cell number and the viability (%) are listed on the lot‑specific Certificate of Analysis (CoA).
Only the expected number of viable cells is reported; the total number of cells (viable + dead) is not specified.
Example:
If the CoA states 600,000 viable cells with 80% viability, the vial originally contained ~750,000 total cells, of which 600,000 were viable after thawing under PromoCell QC conditions.
No viability calculation by the user is required when the recommended thawing protocol is followed.
 
Calculating the optimal plating density
Use the number of viable cells stated on the CoA. Apply the recommended plating density for the specific cell type (e.g. 5,000-10,000 cells/cm²).
Example:
600,000 viable cells can be plated:
- into one T75 flask (~8,000 cells/cm²), or
- into one T75 + one T25 flask (~6,000 cells/cm²)

Upon arrival, PromoCell melanocytes can either be thawed immediately and seeded into a tissue culture vessel or stored in liquid nitrogen until needed.

Re‑freezing after subculture
It is technically possible to re‑freeze normal human cells, including melanocytes. However, PromoCell does not recommend repeated freezing, as each freeze-thaw cycle reduces the cells’ proliferation potential.

Recommendation
If freezing after subculture is required:

• Use PromoCell Cryo‑SFM Plus (C‑29920) as the freezing medium.
• Freeze the cells at an early passage, as this helps preserve viability and growth potential.

Baculovirus expression systems are commonly used in combination with insect cell lines (e.g. Sf‑9 or Sf‑21) to produce recombinant proteins, such as cytokines or growth factors.
PromoCell does not use recombinant proteins produced in insect cells for its media.
This applies to 

• Specialized Media for Primary Human Cells
• Blood and Stem Cell Media
• Cancer Cell Media
• Cryo‑SFM Plus Freezing Medium

None of these products contain components produced using a baculovirus/insect cell expression system.
 

PromoCell cell pellets (C‑14***) can be stored under the following conditions:

• -20°C: Can be stored indefinitely
• 4°C: Stable for up to 1 month
• Room temperature: Stable for up to 1 week

Important note
Cell pellets are supplied in RNAlater^TM and do not freeze at -20°C.

PromoCell human mesenchymal stem cells (MSCs) and human pericytes are cryopreserved at the end of secondary culture (passage 1, P1).
After thawing, the cells are therefore in passage 2 (P2).

When human mesenchymal stem cells (hMSC) are differentiated using PromoCell Mesenchymal Stem Cell Neurogenic Differentiation Medium (C‑28015):

• Approximately 90–100% of the cells adopt a neuronal‑like morphology.
• About 60–80% of the differentiated cells are positive for Nissl bodies, as confirmed by Nissl staining.

These results indicate a high differentiation efficiency at the morphological level, with a substantial proportion of cells also showing neuronal cytological features.

PromoCell human adipose‑derived mesenchymal stem cells (MSC‑AT, C‑12977) are characterized according to established criteria.
 
Characterization criteria
1. Trilineage differentiation potential
MSC‑AT are tested for their ability to differentiate into:

• Chondrocytes
• Adipocytes (fat cells)
• Osteoblasts (bone cells)

2. Surface marker expression (flow cytometry)
In accordance with the International Society for Cellular Therapy (ISCT) guidelines, MSC‑AT show:

• Positive expression: CD73, CD90, CD105
• Negative expression: CD14, CD19, CD34, CD45, HLA‑DR

This combination of functional and phenotypic criteria confirms the identity and quality of the MSC‑AT population.

Successful transfection of endothelial cells depends on several factors, including cell viability and density, the transfection reagent, the type and quality of the nucleic acid (e.g. plasmids, siRNA, oligonucleotides), and the culture medium and supplements used.

Recommended medium for transfection
PromoCell recommends using heparin‑free culture conditions during the transfection step, as heparin can reduce transfection efficiency.

• Preferred option: Endothelial Cell Growth Medium MV2 (C‑22022 / C‑22121), which is heparin‑free
• Alternative option: Use one of the following Growth Medium Kits without adding the ECGS/heparin supplement to the basal medium:
  • Endothelial Cell Growth Medium Kit (C‑22110)
  • Endothelial Cell Growth Medium 2 Kit (C‑22111)
  • Endothelial Cell Growth Medium MV Kit (C‑22120)

Important note
Before and after transfection, endothelial cells should be cultured in complete Growth Medium including heparin to ensure optimal cell growth and recovery.