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Q: What are the unit sizes of the cells?
A: It depends, we usually provide 0.5M, 1M, 5M, or 10M on most of our cryopreserved cells.
Q: Do you have a Safety Data Sheet or risk group/biosafety level information that you can provide me?
A: We have relevant MSDS documents. Please fill out the inquiry form or send an email to [email protected] to request the needed documents.
Q: Which QC tests are performed on?
- 1.Sterility: Negative for contamination that includes bacterial, fungal, Mycoplasma (PCR), and negative for human pathogens;
- 2.Viability: >95% post-thaw viability;
- 3.Identity: Using Short Tandem Repeat (STR) analysis, 16 probes (15 STR loci plus Amelogenin) are checked;
- 4.Growth: based on the morphology, immunocytochemistry of multiple undifferentiation markers.
Q: What should be done with the receiving cells?
A: Immediately after receiving the package, transfer the cryopreserved cells from the dry ice shipping container to liquid nitrogen to prevent the cells from thawing. Do NOT store cells at -20℃ or -80℃.
Q: How do I recover cryopreserved cells?
- 1.Remove a vial of cells from liquid nitrogen, taking care to protect your hands and eyes.
- 2.Loosen the cap on the vial for 1/4 second to release the liquid nitrogen that may be trapped in the threads, then re-tighten the cap.
- 3.Place the frozen vial in a 37℃ water bath. Gently hold and rotate the vial until the contents are completely melted. The vial was immediately removed from the water bath, wiped clean with 70% ethanol, and then transferred to a sterile area.
- 4.Carefully remove the cover without touching the internal threads. Gently resuspend the contents of the vial and dispense into a balanced poly-L-lysine or fibronectin-coated culture vessel containing the culture medium (see specific instructions listed on the product table).
- 5.Close the lid or lid of the culture dish and gently shake the culture dish to evenly distribute the cells. If necessary, loosen the cover for gas exchange.
- 6.Return the petri dish to the incubator (37℃, 5% CO2 / 95% air).
- For best results, do NOT disturb the culture for at least 16 hours after starting the culture. The medium was renewed the next day to remove residual DMSO and unattached cells (unless stated otherwise on the product table). Note: For detailed instructions, see the cell manual.
Q: Do I need to centrifuge the cells to remove DMSO when recovering cryopreserved cells for the first time?
A: We do NOT recommend centrifuging the cells to remove DMSO residue.
Q: How to prevent cell contamination?
- 1.When the cells are thawed in a water bath, the nozzle of the cryopreservation tube should be higher than the water surface to prevent the liquid contaminating the cells.
- 2.Water baths are prone to bacterial growth, and water should be disinfected regularly to avoid contaminating cells.
- 3.When using cell culture dishes, they must be handled aseptically to prevent cell contamination.
Q: How much volume of media should be added to the cell culture flask?
A: We recommend adding 5ml of medium to the T-25 flask, 15ml of medium to the T-75 flask, and 30ml of medium to the T-150 flask.
Q: How often should I change the cell culture medium?
A: For specific instructions, see the product table for the type of battery used. Generally, the medium should be changed every 2-3 days depending on the confluence of cells.
Q: At what confluence should I pass cells?
A: It depends on the cell type, refer to the manual for details.
Q: (For weak adherent cell lines) These cells do not show strong adhesion. When we add the PBS into the flask, these cells are very easy to fall. How to subculture?
A: Cells do show weak adhesion as compared to most other adherent cell lines in our hands. Please take great care when you trypsinize cells or replace the medium. Normally, you can use diluted trypsin (e.g. 0.01%), which is sufficient to detach cells.
Q: What is the difference between primary cells and cell lines?
A: Primary Cells – Cells derived directly from human or animal tissue. And primary cells usually have a limited lifespan. Compared to cell lines, primary cells maintain a lot of characters and markers seen with the donors.
Cell Lines – Cells have undergone a genetic transformation that has been continually passaged over a long period of time. Cell lines are finite or continuous. Usually, a cell line can be sub-cultured for 30-80 passages. Compared to primary cells, cell lines have lost the true characteristics of the original tissue from which they were isolated but are preferably used for convenience as they are easy to handle and widely published.
Q: How to determine cell type?
A: Actually, cell type can be confirmed in multiple ways. We usually take the morphology of cells as one of the most important ways to identify the correct cells. Besides, we also use immunofluorescence to confirm the presence of identifying markers for some of the cell types.
Q: Can I get donor information for cells?
A: If you have specific requirements (age and/or gender), please contact our customer service department before ordering.
Q: Will my primary cell vial be 100% pure?
A: Primary cells are never 100% pure, but we try to make the cells as pure as possible.
Q: What passage number would endothelial primary cells be?
A: You will receive endothelial cells from passages 2 or 3, depending on cell purity.
Q: How many passages can a normal primary cell take?
A: It depends on the cell type, refer to the manual for details. Primary endothelial cells can be expanded 3-7 passages by the split ratio of 1:2 under the cell culture conditions specified by AcceGen.
Q: Can non-proliferating cells be sub-cultured?
A: Non-proliferating cells cannot be sub-cultured because they do not proliferate. This includes the following cell types: neurons, microglia, macrophages, and some other cell types. See the instructions for details.
Q: Do we perform human leukocyte antigen typing for cells?
A: If you have specific requirements, please contact our customer service department before ordering.
Q: How do I choose a primary cell culture environment?
A: We recommend using 37℃, 5% CO2 incubator to culture all primary cells of AcceGen.
Q: Can I refreeze AcceGen primary cells?
A: We do NOT recommend customers to refreeze AcceGen primary cells.
Q: As for blood collection, what is the difference between K2EDTA, Sodium Citrate and Sodium Heparin?
A: The tubes may contain additional substances called anticoagulants, which can be helpful for better preservation of the blood for further processing in the medical laboratory. The choice of different anticoagulants depends upon the downstream medical/biochemical analysis being undertaken.
- ◇Heparin is a highly charged biological molecule-polymeric glucosaminoglycan. It prevents coagulation of blood but can be co-purified with the nucleic acids depending upon the preparation method used. It is a well-known inhibitor of PCR and preferably should be avoided if collecting blood for PCR analysis.
- ◇EDTA can be used for whole blood hematology determinations and immunohematology testing like ABO grouping, Rh typing, antibody screening.
- ◇Citrate can be used in different types of blood bank studies that include HLA phenotyping, DNA and paternity testing.
Q: Do you have any primary epithelial cells from main organs like lung, liver, colon, or kidney that are HLA-A2 positive? If not, what other primary cells that are NOT immune cells you have that are HLA-A2 positive?
A: We do not have primary epithelial cells from organs. Currently, only immune cells and PBMCs have HLA information.
Q: Would you be able to provide some additional information on tight junction protein expression- E-cadherin, ZO-1 or Claudin 3 in human intestinal epithelial cells? How long do these cells take to differentiate and form tight junction proteins?
A: Epithelial Cells grow in the medium form a monolayer of polarized epithelial cells with tight junction formation as evidenced by ZO-1 (tight junction marker) staining.
ZO-1 staining is typically performed on day 3 or 4. A time course of expression has not performed.
Q: Comparing to primary cells directly originate from organs, are there any differences in iPSC derived primary cells?
A: iPSCs can be differentiated to functional and specific cells in vitro with demonstrable in vitro and in vivo research. Genetic modifications employed at the iPSC level can deliver desirable immunotherapeutic attributes to the generated effectors.
Q: Can iPSC cells be grown without a feeder layer? If a feeder layer is required are you using MEF cells?
A: iPSCs usually grow best when attached to other cells or an extracellular matrix and grow traditionally in cultures with feeder layers.
MEFs are fairly universally used. If using MEFs as a feeder layer, the MEF media must be removed before the iPS cells can be plated. The iPS cells can be cultured in MEF dishes conditioned with iPS medium. We recommend conditioning new feeder dishes prior to passaging iPS cells into them.
Q: What is the minimum quantity you can provide for miRNA Agomir/Antagomir service?
A: The minimum quantity is 2 OD (1 OD corresponds to 33μg). Please note we have significant bulk order discounts.
Q: What kinds of labels you can do for miRNA Agomir/Antagomir service?
A: We usually can label the Agomir/Antagomir with cholesterol or fluorescent tag, such as FAM, CY3, etc.