Technical Library

Recent Entries in Technical Library

• How does PromoCell recommend subculturing the chondrocytes (e.g. from T25 into T75 flasks, or into petri dishes)? Does PromoCell recommend a specific type or brand?

  We recommend a seeding density for chondrocytes between 10,000 and 20,000 cells/cm². This means that a subconfluent T25-flask with approx. 900,000 cells/T25 flask (36,000 cells/cm² ) may be either split into 3 new T25 or seeded in one T75 flask or in one 100 mm petri dish. We do not recommend a specific type or brand for the culture of HCH.

  Related Links and Documents

  • HCH
• What culture conditions are required for culturing PromoCell Normal Human Cells in the respective PromoCell media?

  PromoCell Normal Human Cells should be cultured in the appropriate medium at 37°C and 5% CO₂ in a humidified atmosphere.
  Please note: If using cell culture flasks w/o filter cap, unscrew the cap by half a turn to allow sufficient ventilation.

• Can you please provide me a protocol for the expansion of CD34+ Progenitor Cells in PromoCell Expansion Medium XF?

  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.

  Related Links and Documents

  • App Note: Expansion of primitive Hematopoietic Progenitor Cells
  • Hematopoietic Stem Cell Culture
• Why does the macrophage differentiation in the PromoCell protocol take 10 days? In the literature or following the feedback from other researchers, it usually only takes 7 days.

  M1 / M2 polarization also takes seven days in the PromoCell system but the protocol contains two more days for optional macrophage activation. If you only want non-activated M1 / M2 macrophages, the process is usually completed after 7 days.
  Nevertheless, PromoCell does not recommend shortening the 10-day protocol because you actually get a plus in viability and cell yield (due to the re-attachment of floating cells) on day 8-10 due to the media change.

  Related Links and Documents

  • Application Note: Differentiation of M1- or M2-Macrophages
  • M1-Macrophage Generation Medium DXF
  • M2-Macrophage Generation Medium DXF
• How can I avoid precipitates when preparing my Mesenchymal Stem Cell Adipogenic Differentiation Medium 2?

  The supplement should be at room temperature when added to the MSC Adipogenic Basal Medium 2. It may also be beneficial to invert the tube a few times to bring precipitates back into solution.

  Please note: It is not recommended to filter the basal medium, supplements, or complete medium, as components that induce or promote differentiation may be removed, resulting in a low differentiation rate when using the medium.

  Related Links and Documents

  • Mesenchymal Stem Cell Adipogenic Differentiation Medium 2
• We use Macrophage Generation Media from Promocell to get differentiated macrophages from fresh PBMC. Would it be okay if the attachment step with the Monocyte Attachment Medium is done overnight instead of 1.5 h?

  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.

  Related Links and Documents

  • Monocyte Attachment Medium
• Could you please let me know whether you have used the baculovirus expression system to produce components of your media?

  Baculovirus is generally used in conjunction with insect cells (Sf-9, Sf-21) to produce recombinant proteins (cytokines, growth factors).
  None of our Specialized Media (Media for Primary Human Cells; Blood and Stem Cell Media, Cancer Cell Media) contain recombinant proteins produced in insect cells. This also applies to our Cryo-SFM Freezing Medium.

• Do I need precoated flasks when growing my MSC in PromoCell Mesenchymal Stem Cell Growth Medium XF (C-28019)?

  Our MSC Growth Medium XF (Ready-to-use) provides a xeno-free culture system for human MSCs. It contains all growth factors and supplements except attachment- and spreading factors. Therefore, culture vessels must be precoated with 10 μg/ml human or bovine fibronectin.
  
  Protocol: Fibronectin coating
  Dilute the Fibronection Solution to 10 μg/ml final concentration in Dulbecco´s PBS w/o Calcium and Magnesium. Overlay the culture surface of your tissue culture vessel with an amount of the diluted Fibronectin Solution sufficient to effectively coat the complete surface. Be sure that the entire surface is covered. Place flasks on a level surface at RT for 60 min. Aspirate the excess Fibronectin Solution and use immediately or let air-dry the open vessel under a laminar flow bench. Unused vessels may be stored at 4°C for up to 2 weeks.

  Related Links and Documents

  • Mesenchymal Stem Cell Growth Medium XF
• Is it normal that CD34+ progenitor cells attach to tissue culture plastic after a week-long culture in HPC Expansion Medium XF (C-28021)?

  The cells sink down to the bottom of the culture vessel but don’t really attach. They retain a roundish morphology and can be rinsed off with culture medium easily.

  Related Links and Documents

  • Human CD34+ Progenitor Cells
  • Hematopoietic Progenitor Expansion Medium XF
• How should I handle PromoCell’s proliferating cells after arrival?

  Short description:
  Unpack the box and place the T25 flask(s) in the incubator for 3 hrs (closed cap). Then check confluency under the microscope.
  When the density is < 70%, aspirate the medium using sterile conditions and add 5-10 ml of the appropriate Growth Medium. The cells should be subcultured according to the subcultivation protocol given in the cells’ Instruction Manual once they have reached > 70 % confluency.

  Related Links and Documents

  • Subcultivation of primary cells
  • DetachKit
  • Accutase Solution