Technical library

The activation of macrophages as such is complete after 24 hrs. However, to maintain the activation status over a longer period of time (i.e., several days), fresh activation factors should be added with every medium change.

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

The difference between PromoCell NHEK and NHEK GM3 lies in the growth medium used during cell isolation:
NHEK (Normal Human Epidermal Keratinocytes) are isolated in Keratinocyte Growth Medium 2 (C‑20011), a serum‑free medium that contains bovine pituitary extract (BPE).
NHEK GM3 are isolated in Keratinocyte Growth Medium 3 (C‑20021), an improved serum‑free and BPE‑free medium, offering a more defined culture environment.
 
Both NHEK and NHEK GM3:

• are isolated from the epidermis of juvenile foreskin or adult skin, and
• are available from single‑donor or pooled‑donor preparations.

Yes. Normal Human Epidermal Keratinocytes isolated in Keratinocyte Growth Medium 2 (NHEK‑GM2; C‑12001, C‑12003, C‑12005, C‑12006) also grow in PromoCell Keratinocyte Growth Medium 3 (C‑20021).
When cultured using the protocol with fixed medium‑change intervals, these cells:

• grow slightly faster than in Keratinocyte Growth Medium 2, and
• achieve more than 15 population doublings (PD).

Conversely, NHEK isolated in Keratinocyte Growth Medium 3 (NHEK‑GM3; C‑12011, C‑12013, C‑12015, C‑12016) can also be cultured in Keratinocyte Growth Medium 2 (C‑20011).
When using the classical subcultivation protocol (subculture at 70–90% confluency), these cells:

• grow slightly slower than in Keratinocyte Growth Medium 3, but
• also reach more than 15 population doublings.

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 primary cells are cryopreserved using a computer‑controlled freezing process in the gas phase of liquid nitrogen and subsequently stored in the liquid phase of LN₂.
Cryopreservation in liquid nitrogen is a well‑established method for long‑term storage of primary cells and stem cells. When continuously stored in liquid nitrogen, PromoCell cells can be maintained for more than 10 years without loss of viability.
 
Supporting evidence
Long‑term stability of cryopreserved primary cells has been demonstrated in the literature. For example, Kumar et al. showed that adipose‑derived stem cells stored in liquid nitrogen for approximately 12 years retained viability, stem cell characteristics, differentiation capacity, and regenerative potential.

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.

Yes. PromoCell Normal Human Epidermal Melanocytes derived from juvenile foreskin (NHEM.f, C‑12400) can be cultured in PromoCell Melanocyte Growth Medium M3 (C‑24310).
Under these conditions, NHEM.f can achieve more than 15 population doublings, provided that the recommended culture conditions are followed.