Technical library

PromoCell human uterine microvascular endothelial cells (HUtMEC, C‑12295) are isolated from the middle layer of the uterine wall, the myometrium.
In general, tissue donors are not hormonally pre-treated prior to tissue collection, although individual donor histories may vary.

No. PromoCell human cardiac microvascular endothelial cells (HCMEC) are not endocardial cells.

• Endocardial endothelial cells line the endocardium, the innermost layer of the heart that covers the cardiac chambers and ventricles.
• PromoCell HCMECs are isolated from the capillaries within the heart muscle (myocardium).

Because these cells originate from small blood vessels (capillaries) rather than from the endocardium, they are correctly referred to as microvascular endothelial cells.

PromoCell offers two different DC Generation Media, optimized for different starting materials.
 
Freshly isolated mononuclear cells (MNC) or monocytes
For freshly isolated MNC or monocytes, PromoCell recommends DC Generation Medium XF (C‑28052). This serum‑free and xeno‑free medium is optimized for the direct differentiation of monocytes into immature and mature myeloid dendritic cells.

Cryopreserved monocytes
For cryopreserved monocytes, PromoCell recommends DC Generation Medium (C‑28050).
This medium is optimized to support dendritic cell generation after thawing of monocytes.
 
Important note for fresh MNC with DC Generation Medium (C‑28050)
If DC Generation Medium (C‑28050) is used with freshly isolated MNC, an additional initial step is required:
Monocyte Attachment Medium (C‑28051) must be used first to ensure efficient adherence and enrichment of the monocyte fraction.
 
Further details are provided in the Instruction Manual and the corresponding Application Note: Generation of monocyte-derived Dendritic Cells (moDCs).

No. PromoCell does not recommend a specific brand of cell culture plasticware. Standard tissue culture-treated (TC-treated) plasticware is suitable for dendritic cell generation from peripheral blood monocytes.

No significant difference is observed in the percentage of monocytes that differentiate into dendritic cells (DCs) when comparing freshly isolated and cryopreserved monocytes when using PromoCell DC Generation Medium (C‑28050).
However, there is an important practical difference to consider:

• Higher initial cell loss with cryopreserved monocytes compared to when fresh monocytes are used.
• Impact on final number of differentiated cells

As a result, while the differentiation efficiency itself is comparable, the absolute number of differentiated DCs obtained at the end of the culture will usually be higher when starting from fresh monocytes.

No. An overnight attachment step is not recommended. The PromoCell Monocyte Attachment Medium is specifically formulated for short‑term attachment of monocytes and does not provide sufficient nutrients or survival factors for extended incubation.
 
Why overnight incubation should be avoided

• Induction of apoptosis: Leaving PBMCs in Monocyte Attachment Medium for longer than 1.5–2 hours will induce apoptosis of monocytes, leading to significant cell loss.
• Reduced purity due to lymphocyte attachment: Lymphocytes survive longer under these conditions. During prolonged incubation, some lymphocytes may also attach and can no longer be removed by washing, resulting in reduced monocyte purity.
• Negative impact on macrophage differentiation: The combination of monocyte loss and lymphocyte contamination compromises downstream differentiation into macrophages and affects reproducibility.

For these reasons, overnight incubation in Monocyte Attachment Medium is inappropriate.

Recommended alternatives

• Standard protocol: 1.5 hours monocyte attachment
• If timing is critical: The attachment step can be shortened to 1 hour

Best practice: 
- Pre‑equilibrate the Monocyte Attachment Medium in the incubator
- Add the appropriate amount of PBMC suspension directly
- Under these conditions, most monocytes are already attached after 1 hour

Monocyte enrichment by adherence selection using PromoCell Monocyte Attachment Medium (C‑28051) offers several practical and biological advantages:

Rapid enrichment
Large numbers of adherent monocytes can be obtained within approximately 1.5 hours, making this method very fast compared to bead‑based separation techniques.
High purity
When washing steps are performed correctly, a monocyte purity of 80–90 % can be achieved.
“Untouched” monocytes
This method does not involve magnetic microbeads. Therefore, monocytes are not exposed to bead binding or internalization, resulting in untouched cells with preserved physiological properties.
Improved cellular health
The absence of magnetic beads also prevents phagocytosis of microbeads by monocytes, which can negatively affect cell viability and function.
Time‑saving and cost‑effective
Compared to immunomagnetic separation, adherence‑based enrichment is less labor‑intensive, faster, and more economical, while still delivering reliable results.
 

Several established methods are available for the detection of mycoplasma contamination in cell cultures, including:

• Culture‑based methods: Growth on agar plates or in liquid/semi‑solid media
• Microscopy‑based methods: DNA staining (e.g. DAPI)
• Immunological methods: Detection using mycoplasma‑specific antibodies
• Biochemical assays
• PCR‑based assays

Recommended approach
PCR‑based mycoplasma detection is widely used because it:

• Is highly sensitive
• Detects all mycoplasma species commonly found in cell culture
• Delivers results within 3-5 hours

 

No. Mycoplasma contamination cannot be detected with the naked eye and cannot be visualized by standard light microscopy.
Mycoplasmas are extremely small and can only be directly visualized using electron microscopy. For routine and highly sensitive detection, PCR‑based mycoplasma assays are recommended.

PromoCell uses a classification system similar to, but not identical with, the Fitzpatrick Skin Classification.

The Fitzpatrick classification distinguishes six phototypes (I–VI) based on skin pigmentation (melanin content) and the skin’s response to sun exposure:

• Phototype I: Very fair skin, always burns, never tans
• Phototype II: Fair skin, burns easily, tans poorly
• Phototype III: Medium skin, tans after initial burn
• Phototype IV: Light brown skin, rarely burns, tans easily
• Phototype V: Brown skin, very rarely burns, tans darkly
• Phototype VI: Dark brown or black skin, never burns

PromoCell donor classification:
For PromoCell skin tissue donors, information is available on:

• Skin color (white, brown, or black)
• Eye color
• Hair color

However, no information is available on individual tanning or burning behavior.
Therefore, PromoCell classifies donors into the following three phototype groups:

• Light (corresponding to Fitzpatrick phototypes I and II)
• Moderate (corresponding to Fitzpatrick phototypes III and IV)
• Dark (corresponding to Fitzpatrick phototypes V and VI)

Information on the phototype group is available for most cell lots derived from juvenile or adult skin tissue. Our Scientific Support team can assist in the selection of suitable cell lots based on the desired donor phototype.