Technical library

Our hMSC-AT are characterized by their differentiation potential into chondrocytes, fat cells, and bone cells. In addition, we determine the presence of CD73, CD90 and CD105 expression as well as the absence of CD14, CD19, CD34, CD45 & HLA-DR expression by flow cytometry as proposed by the International Society for Cellular Therapy.

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.

It is not necessary to use coated flasks for (most of) our Normal Human Cells but it can be done. As coating with extracellular matrix proteins can affect cellular metabolism, it is recommended to use the same coating material for a complete set of experiments.

Cells that need to be grown on coated dishes:

• Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need FN- or VN-, or Collagen type I-coating (for details, please view the Product Manuals) when grown in PromoCell MSC Growth Medium XF (C-28019) and/or when being differentiated in our MSC Neurogenic (C-28015), Adipogenic (C-28016), or Osteogenic (C-28013) Differentiation Media.
• Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin- or Vitronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055, C-28056).
• For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020), the TC plates should be pre-coated with collagen type I.

During our quality control we do not determine the presence or maintenance of respective stem cell markers in the tumorspheres. We use a functional approach instead, by culturing the tumorspheres over serial passages in C-28070. Tumorsphere formation requires the biological features of Anoikis resistance and self-renewal and therefore indicates the presence of cancer stem cells (CSC)/cancer initiating cells (CIC) in the spheres. 3D tumorsphere culture thus allows scientists to study the biology of CSCs without any background knowledge on CSC markers.

The abbreviations hMDM‑GMCSF(-) and hMDM‑MCSF(-) describe the origin, polarization, and activation state of the cryopreserved macrophages.
 
Meaning of the abbreviations
hMDM‑GMCSF(-):
hMDM: human monocyte‑derived macrophages
GM‑CSF: cells were differentiated (polarized) using GM‑CSF
(-): cells are not activated
→ These cells represent non‑activated M1‑polarized macrophages
hMDM‑MCSF(-):
hMDM: human monocyte‑derived macrophages
M‑CSF: cells were differentiated (polarized) using M‑CSF
(-): cells are not activated
→ These cells represent non‑activated M2‑polarized macrophages
 
Culture and activation

• Seed the macrophages in fibronectin‑coated tissue culture vessels
• After plating, they form biologically functional adherent cultures and can be maintained for several weeks
• Optional, user‑defined activation can be performed after seeding, depending on the experimental setup

Further details on polarization and activation options are provided in the Application Note "Standardized culture of assay-ready and fully functional human primary macrophages".

The following cell lines have been

tested at PromoCell

to form tumorspheres in 3D Tumorsphere Medium XF (C-28070):

• U-87 MG
• MCF-7
• MDA-MB-231
• HT-29
• HT1080
• HepG2
• A-549
• Panc-1
• LNCaP
• A-431

⇒ For more details, please view the attached

Application Note

. In addition, we have received

customer feedbacks

for the following cell lines:

• HCT-116 (human colorectal carcinoma cell line)
• Capan-1 (human pancreatic adenocarcinoma cell line)
• PC3 (human prostate cancer cell line)
• C42B (osteotropic prostate cancer cell line)
• NCI-H23 (human lung epithelial adenocarcinoma cells)
• IMR-32 (human neuroblast cell line)
• A818-6 (human pancreatic ductal adenocarcinoma cell line)
• HEK293 (human embryonic kidney cells)
• Calu-1 (non-small-cell lung cancer cell line)

Yes, we have received a customer feedback that our MSC Growth Medium 2 also works for rat MSCs. The rat cells grow nicely in this medium and have a good viability.

• HDLEC and HDBEC both express the typical endothelial cell marker CD31 (= PECAM-1).
• HDLEC cultures are additionally tested positive for podoplanin, a transmembrane glycoprotein involved in lymphatic vessel formation, whereas HDBECs are podoplanin-negative.

Yes, we have received a customer feedback that our 3D Tumorsphere Medium XF (C-28070) has been successfully used for tumorsphere formation of mouse cell lines.

It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured, estrogen-responsive cells (Berthois et al., 1986). Furthermore, phenol red can also interfere with some analytical methods like photometric analyses.

Further Information

Berthois et al.; Proc Natl Acad Sci U S A. 1986 Apr;83(8):2496-500