Technical library - RESOURCES

Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.

The NHEK (primary human keratinocytes) are isolated in our serum-free Keratinocyte Growth Medium 2 (C-20011), the NHEK GM3 in our improved serum-free and BPE-free Keratinocyte Growth Medium 3 (C-20021). Both, NHEK and NHEK GM3 are available from single or from pooled donors isolated from the epidermis of juvenile foreskin or adult skin.

Yes, the NHEK-GM2 (C-12001, C-12003, C-12005, C-12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C-20021). Using the protocol with the fixed intervals, they grow slightly faster than in Keratinocyte GM2 and proliferate for > 15 PDs. Conversely, NHEK-GM3 (C-12011, C-12013, C-12015, C-12016) also grow in the existing Keratinocyte Growth Medium 2 (C-20011). When using the classical subcultivation protocol (density > 70% - 90%), they grow slightly slower compared to Keratinocyte GM3, but also reach > 15 PDs.

The MSCs attach even without fibronectin coating, but the risk of detachment during differentiation is much higher. We therefore strongly recommend using fibronectin- or vitronectin-coated TC vessels.

PBMCs (= peripheral blood mononuclear cells) consist mainly of lymphocytes and monocytes. Cryopreservation causes the CD14+ monocytes to significantly lose their ability to attach to TC plastic. Therefore, the use of frozen PBMCs as starting material for DC generation is not possible because the purification step with Monocyte Attachment Medium will not work. You can either start with cryopreserved CD14+ monocytes (C-12909) or use freshly isolated mononuclear cells or fresh CD14+ monocytes. Please refer to the Product Manual of C-28050 or our Application Note [Generation of monocyte-derived Dendritic Cells] for the appropriate protocol.

For our HNEpC and HTEpC, please refer to the instructions in the AppNote describing the differentiation of these cell types using our ALI medium.
We do not provide instructions for HSAEpC because our medium is not suitable for differentiation of this cell type at the air-liquid interface.
For our HBEpC we have special ALI prescreened lots in stock that were successfully tested for barrier function in our QC. Please contact our technical customer support for more information.

PromoCell cells are frozen down in the gas phase of liquid nitrogen (computer-controlled freezing machine) and then stored in the liquid phase of LN2. The cryopreservation in LN2 is an acknowledged method for long-term storage of primary cells and stem cells. When stored in liquid nitrogen, the cells can be maintained for a period of > 10 years without affecting viability. For example, Kumar et al. showed that adipose-derived stem cells stored in LN2 for about 12 years still retained their regenerative potential, stem cell property, viability as well as differentiation ability.

Further Information

Kumar et al.; Exp Eye Res . 2019 Dec;189:107860

We have never tested the cultivation of our assay-ready macrophages in MEM alpha + FBS. We cannot predict whether it will work and therefore strongly recommend the use of our M1 Macrophage Generation Medium XF and fibronectin-coated vessels.

Short protocol:

Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells

Spin down for 10 min at 240xg, aspirate the supernatant, resuspend the pellet at 20,000 cells/ml HPC Expansion Medium XF Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂

Then double the media volume by adding fresh complete medium, e.g., 4 ml suspension culture + 4 ml fresh medium (= 8 ml)

Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days

Example partial medium change:
For a culture volume of 8 ml, spin down the cells, aspirate and discard 4 ml of the supernatant, resuspend the cells and add 12 ml of fresh complete medium (= 16 ml). In combination with the Cytokine Mix E, the HPC Expansion Medium XF typically promotes a 300-1,000 fold expansion of the total cell population. After 2 weeks of expansion about 20-30% of the population express CD34+, indicating a 50-200 fold expansion of CD34+ progenitor cells.

We strongly advise against overnight incubation. The Monocyte Attachment Medium does not contain cytokines/survival factors for the monocytes. If the cells remain in the Monocyte Attachment Medium for longer than 2 hours, they will go into apoptosis and die. The lymphocytes will survive longer. Some of them will attach after such a long time and then cannot be washed off. Therefore, overnight attachment is absolutely inappropriate. However, if time is short, monocyte attachment can be shortened to 1 hour. To do this, it is best to add Monocyte Attachment Medium to the culture vessels in advance and equilibrate it in the incubator. Remove the vessels from the incubator only briefly to add the appropriate amount of concentrated PBMC suspension. Then, even after 1 hour, most of the monocytes are attached.

Yes, our NHEM.f will also grow in our optimized Melanocyte Growth Medium M3 (C-24310) and can achieve > 15 population doublings.

We recommend to use Human Serum AB „off-the-clot”.