Have a question about one of our products? Check out our technical library for recently asked questions from other scientists around the world.
Technical Library
Recent Entries in Technical Library
284 Entries on 6 Pages
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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).
Depending on the donor a total expansion factor of approximately 300-1,000 fold (CD34⁺ plus CD34– cells) and an expansion of the CD34⁺ population by approx. 50–200 fold can be expected when using complete HPC Expansion Medium XF with Cytokine Mix E.
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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.
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Can I use a serum-free medium as negative control in the chondrogenic MSC differentiation? I would like to replace the recommended DMEM+10% FCS.
You could probably use our MSC Chondrogenic Differentiation Medium without inducers (C-28014). It is also serum-free and the same as C-28012, just without chondrogenic inducers.
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Is it possible to thaw and re-freeze the Cytokine Mix M1 (C-39894) and M2 (C-39895)?
The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.
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Can I use a bead bath to thaw PromoCell normal human cells?
Based on negative feedback we have received from customers using bead baths, we strongly discourage the use of bead baths to thaw our cells. It can lead to reduced viability or significantly slower growing cells.
If you don’t have a “normal” water bath but only a bead bath in your lab, thaw the vial in a beaker of water in the bead bath. Ensure the water is heated to exactly 37°C using a thermometer placed in the warmed water. Be sure to hold the vial in your hand, and not in a floater, as described in the thawing protocol.
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Do you find that the domes spread a lot on the Nunclon Sphera plates? Do they remain attached for the entire 3-4 week culture period?
To avoid the spreading of the matrix and to have nice drop-like domes, it is crucial to preheat the plate for 2h at 37°C and to work fast when transferring the gel to the wells. Placing a warming plate under your laminar air flow hood when transferring the gel-cell-mix to the preheated plate may help.
From our testing, we found that Nunclon Sphera wells were not ideal for our organoid system as the dome did not stay adhered, but it was able to move on the bottom and the dome became misshaped overtime. However, the altered shape of the dome did not affect the organoids and were successfully cultured for 4 weeks. If movement is not an issue for the user, we recommend that extra caution be taken during media changes by holding the plate at an angle and carefully pipette to remove old medium. Aspirating with vacuum suction can damage or destroy the dome.
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How do you set up high throughput systems with organoids? Only in 96 well?
It is possible to culture the airway organoids in 96-well U-Bottom plates for suspension cells (e.g., Greiner Bio-One # 650185).
A detailed protocol for the use of 96-well plates can be found in our AppNote. We have not tested 384-well plates or other commercially available plates.Related Links and Documents
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Why use ROCKi for organoid formation?
ROCKi is known to enhance the proliferation of epithelial cells or keratinocyte progenitors and can improve cell survival via conditional reprogramming and the effect is reversible. It is also known to enhance the seeding efficiency on plastic.
This ROCKi conditional reprogramming has also been observed in airway EpCs and cultured organoids:
For our human airway organoids, the use of Y-27632 ROCKi is optional as organoids can still form without it. However, the 3D system has some potential pitfalls like choosing the right plastic or the right ECM. The use of ROCKi may help to keep the system more robust.
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Can you control size / size distribution of organoids?
Possibly. The size of the organoids may depend on the access to nutrients in the gel. For reference, https://pubmed.ncbi.nlm.nih.gov/31173716/
For best results we recommend the following:
- Avoid nutrient gradients. We recommend using a small, drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
- For optimal cell distribution, it is important to control the “gelling” or setting of the ECM gel. Therefore, the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time, otherwise the cells in the BME would sink to the bottom.
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Can you drive organoid orientation (inward vs outward)?
Yes. The orientation can be triggered by the use or lack thereof of ECM.
Without the use of an ECM, the organoids will have a higher outward oriented ratio. You can find a protocol here https://pubmed.ncbi.nlm.nih.gov/30811997/, where cells were first embedded in ECM gel and afterwards ECM was dissociated, and free organoids were re-seeded in suspension without an ECM.
* Please note that we have not tested this method in our labs and thus cannot guarantee it.
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For which cytokeratin are the keratinocytes tested?
PromoCell is using a pan-cytokeratin antibody rather than a specific type of cytokeratin for the quality control of our keratinocytes.
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Why is the actual cell count in the vial at PromoCell sometimes significantly higher than the 500,000 cells stated on the website?
We guarantee > 500,000 viable cells per vial. To do this, we need to freeze more than 500,000 cells, as we do not yet know the viability after thawing at the time of freezing.
For technical/organizational reasons, the initial cell number may also vary from lot to lot, so 2 lots with the same viability may not necessarily contain the same number of cells.Related Links and Documents
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Can your Macrophage Detachment Solution also be used for bone marrow-derived macrophages from mice?
Yes, there is a reference (Campuzano et al.; J Immunol. 2020 Jun 15;204(12):3296) where bone marrow-derived macrophages from mouse were detached using our Macrophage Detachment Solution (40 minutes at 4°C).
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Are your NHEK from the epidermis of the juvenile foreskin mucosal or cutaneous keratinocytes or a mixed population?
PromoCell’s Normal Human Epidermal Keratinocytes (NHEK) from juvenile donors are isolated from both the epidermis of the outer and mucosal (i.e. inner) layers of the foreskin. Thus, each vial contains a mixture of mucosal and cutaneous keratinocytes.
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Why is PBS added in the last step of the Alizarin Red staining protocol (“Osteogenic differentiation and analysis of MSC”)? Should the PBS be aspirated before analysis?
The PBS buffering enhances the Alizarin Red staining (precipitation of the dye) and makes it more intense.
Leave the PBS on the cells after staining/washing and analyze the sample immediately, as the dye may bleed upon prolonged storage without embedding.Related Links and Documents
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What are the disadvantages of the prophylactic use of antibiotics in cell culture?
The use of antibiotics creates a false sense of security and allows users to develop poor aseptic techniques. This leads to low-level contamination with partially resistant bacteria occurring but being overlooked for a time. This then leads to cells with undetected contamination being cultured for extended periods of time, increasing the risk that contamination will spread throughout the laboratory and eventually antibiotic-resistant strains of bacteria may develop.
Mycoplasma infections can also occur more easily, as they are often introduced along with contaminants such as bacteria and fungi.
Last but not least, antibiotics are known to have negative effects on the metabolism of eukaryotic cells – more details on this topic can be found in our blog article “Antibiotics in cell culture: friend or enemy”.Related Links and Documents
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I have accidently stored the Cryo-SFM at -20°C. Can the product still be used in this case?
According to the product manual, Cryo-SFM should be stored at 4-8°C. However, since this solution is used to freeze cells in liquid nitrogen, we assume that storing Cryo-SFM once at -20°C should not have a negative impact on the product quality. After thawing, please store it at 4-8°C as recommended.
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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.
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Are there any differences in the cultivation protocol when cultivating MSCs compared to other cell types?
Yes, there are a few differences:
– We recommend replacing the MSC Growth Medium XF (C-28019) 3-4 h after seeding, as opposed to 16-24 hours after seeding for most other cell types/growth media.
– When MSC Growth Medium XF, MSC Neurogenic Differentiation Medium, MSC Adipogenic Differentiation Medium 2 or MSC Osteogenic Differentiation Medium are used, flasks have to be coated with 10 µg/cm² (human or bovine) fibronectin according to the instruction manual.
– We strongly recommend using Accutase (C-41310) for cell detachment instead of Trypsin. If Trypsin is used, contact time should not exceed 2 min.Related Links and Documents
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After thawing the supplements, I see some precipitation. Is this normal?
Light flocculation may be seen upon thawing the supplements containing ECGS/heparin or BPE. This does not affect the activity of our media.
Optionally, the precipitate can be removed by centrifugation under sterile conditions.We recommend to thaw the supplements (SupplementMix or SupplementPack) at 15-25°C.
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We are working with PromoCell MSCs and MSC Adipogenic Differentiation Medium. Do you have any information on a comparison between Sudan III and Oil Red O and if Sudan III is more efficient at labeling lipid vesicles?
You can also use Oil Red O to stain lipid droplets. At PromoCell, we used to use Oil Red O as well, but switched to Sudan III some time ago for organizational reasons.
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Are PromoCell HPAEC and HPASMC obtained from the proximal or distal part of the pulmonary artery?
Our HPAEC and HPASMC are isolated from the proximal pulmonary artery.
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Why are PromoCell products “for in vitro research use only”?
The basis for the intended use of our products is defined in our Terms & Conditions under the chapter “Use of Goods”.
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How long do I need to activate my macrophages? Can I stop adding activation factors after 24 hours?
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.
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Can the Cytokine Mix E for the Hematopoietic Progenitor Expansion Medium XF (C-39891; 5 ml) be safely aliquoted?
Yes, it is possible to aliquot the 5 ml Cytokine Mix E (C-39891) into 5 x 1 ml.
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What is the difference between PromoCell NHEK and NHEK GM3?
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.
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Will my NHEK (isolated in PromoCell Keratinocyte Growth Medium 2) grow in PromoCell Keratinocyte Growth Medium 3?
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.Related Links and Documents
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Will the NHEM.f (C-12400) and NHEM.f M2 (C-12402) also grow in PromoCell Melanocyte Growth Medium M3?
Yes, NHEM.f and NHEM.f M2 also grow in our optimized Melanocyte Growth Medium M3 (C24310) and can achieve > 15 population doublings.
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Which human AB serum does PromoCell recommend to generate M0 macrophages?
We recommend to use Human Serum AB „off-the-clot”.
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I would like to differentiate hMSC into bone cells and further maintain the cells in culture after differentiation. Which medium should I use?
Osteogenic differentiation of hMSCs with PromoCell MSC Osteogenic Differentiation Medium takes ∼12-14 days (please view the respective Application Note for a detailed protocol). Use fibronectin-coated plates and change the medium every third day.
The bone cells tend to detach from the plastic after approximately 2 weeks, when differentiation is complete. Therefore, the tests should be performed promptly and the cells should be maintained in MSC Osteogenic Differentiation Medium until then.
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I am performing MSC chondrogenic, adipogenic, and osteogenic differentiation and am staining the cells. The protocol says to use Saccomanno Fixation Solution. Is there an alternative method to fix the cells because I don’t have Saccomanno Fixation Solution?
Yes, you can use 4.5% neutral buffered formalin. Paraformaldehyde should work as well.
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Is PromoCell’s Cryo-SFM produced under GMP standard?
No, our Freezing Medium Cryo-SFM (C-29910) is not produced under GMP standard. It is for in vitro research use only and not appoved for diagnostic or therapeutic procedures.
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Is it possible to differentiate M1 macrophages from PBMC in 96-well plates?
At PromoCell, we have not tested macrophage differentiation from PBMC in 96-well plates, but we know from users that it is possible.
According to a customer the mononuclear cells differentiate very well in the 96-well format. A plating density of 1 million PBMCs (without prior determination of monocyte content) per well has been shown to be optimal. The working volume in a 96-well plate is usually 100 µl.
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Can the medium for M2 macrophages be switched into RPMI or RPMI + M-CSF after differentiation?
Unfortunately, we did not test this in our hands, and it must be tested by the customer. In fact, our medium is completely different from RPMI and therefore we cannot predict if this is working. We only know the successful long-term culture from our system with our media.
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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
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I differentiated M1 macrophages from self-sourced PBMCs following your differentiating protocol. In the final cell culture, I can see small cells attached to the macrophages which are CD3+ (T-cells). What is the reason for the T-cell contamination after the differentiation?
The reason for the higher number of lymphocytes in the macrophage culture is probably due to an insufficient washing step during the purification of the monocyte via adherence. The 3 washing steps in our protocol are essential to receive a monocyte population of over 90%.
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How long do I have to activate the cryopreserved macrophages to measure cytokine release?
In general, we recommend activating the cells for 24 hours or at least over night for all kind of activations. If the activation is not optimal in your experimental setting, you can increase or decrease the activation time accordingly.
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I would like to analyze the macrophages by microscope and I would therefore have to plate the cells on fibronectin-coated glass. Did you test if the macrophages grow on fibronectin-coated glass?
We did not test if the macrophages attach on fibronectin-coated glass.
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Following the recommended seeding density for your M1 macrophages (100,000 cells per cm2), I would need too many cells to have a complete 96- or 384-well plate. This is too expensive for me, can I reduce the number of cells per cm2?
The recommended seeding density with 100,000 cells per cm2 is needed for a confluent cell layer as the cells do not proliferate.
However, you can reduce the seeding density by the factor 3 to 5 and the macrophages are still viable.Related Links and Documents
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Do you recommend a certain negative control for the activated macrophages to compare the cytokine release with?
Even non-activated macrophages do release a certain amount of cytokines. Furthermore, you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore, we do not think it is possible to have a general negative control for the cytokine release.
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Does PromoCell have any data regarding the cytokine profile and different markers of the M1/M2 macrophages after activation?
No, we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
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Which cytokine concentrations does PromoCell recommend for activating M1/M2 macrophages?
You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.
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Did PromoCell try plating the cryo-macrophages in 96-well plates?
We did not plate the cryo-macrophages in 96-well plates. However, we heard from other customers that they have successfully used our macrophages in this kind of plate.
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I received 3 DetachKits yesterday. Some of the tubes have different colors, although they all come from the same batch.
The components of the PromoCell DetachKit may occasionally arrive with a non-uniform color appearance. This phenomenon is known to PromoCell’s Quality Assurance Dept.
It is reversible and has no influence on the quality of the product.Related Links and Documents
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Are endothelial cells α-SMA-negative under all circumstances?
Scientific findings from different groups, as well as our own results indicate that the presence or absence of α-SMA is not a valid indicator for the composition of a given cell population. Research findings and our inhouse data suggest that endothelial cells are not α-SMA-negative under all circumstances. α-SMA negativity is not an intrinsic property of endothelial cells but can vary depending on extrinsic influences.
Lot-specific values for α-SMA in our Certificates of Analysis (CoAs) are therefore not considered meaningful.
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Is vWF a great marker for Endothelial Cells?
Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (vWF) are more linked to the formation of a confluent endothelial monolayer. Research shows that vWF expression is dynamic and highly dependent on the cell culture conditions such as confluence and passage number.
Therefore, having vWF as a quality control marker for each lot of ECs is not really necessary.Related Links and Documents
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The components of my DetachKit show different colors. Is this normal or does it affect their quality?
The components of the PromoCell DetachKit may arrive on occasion with a non-uniform color appearance. This phenomenon is known by PromoCell’s Quality Assurance. It is reversible and does not influence the quality of the product.
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Can I defrost Accutase Solution, prepare aliquots and refreeze them?
Yes, Accutase Solution can be defrosted, aliquoted, and then refrozen.
Defrosting: Accutase should be defrosted overnight in the refrigerator or placed in a tub of cold tap water. Do not defrost in a 37°C water bath.
Stability: Once thawed, it is stable for at least 2 months in the refrigerator if stored promptly after use.Related Links and Documents
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Is it possible to refreeze the hCD34 progenitor cells after having amplifed them in PromoCell Hemaotopoietic Progenitor Cell Expansion Medium XF?
Yes, it is possible to refreeze them.
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Why are the cryopreserved macrophages abbreviated as hMDM-GMCSF(-) and hMDM-MCSF(-), respectively? What does the (-) stand for?
- hMDM-GMCSF is the abbreviation for human monocyte-derived macrophages. They are polarized [⇒ differentiated with GM-CSF] but non-activated [⇒ (-)] M1 macrophages.
- hMDM-MCSF is the abbreviation for human monocyte-derived polarized [⇒ differentiated with M-CSF] but non-activated [⇒ (-)] M2 macrophages.
The macrophages can be seeded into all kinds of TC vessels. After plating, they can be maintained as biologically functional adherent cultures for several weeks.Optionally, user-customizable activation of the cells can be performed. For details, please see attached Application Note, page 2, Fig. 3 and page 5, Tab. 1).
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