How do you store cells?
The cells are stored in the vapor phase of liquid nitrogen tank.
How do you send out cells?
ALSTEM’s cryopreserved cell products are delivered to your destination in dry ice or Cryoport liquid nitrogen shipping solution (more expensive).
Do you have iPSC licenses?
Yes, we license the technology from iPS Academia Japan, Inc. This licensing agreement enables us to develop, manufacture, and distribute cell products using iPSC technology.
Do you authenticate cells?
We don’t authenticate the cells in house; however, we can send the cells to ATCC for authentication if requested.
Do you check for mycoplasma contamination? If so, how?
Yes, all cell lines we generated will be tested by our MycoDectTM mycoplasma detection kit (Catalog No. MD050). we promise that all of ALSTEM’s cell products are mycoplasma-free.
For the iPS cells, have you done the karyotyping? Have you tested chromosomal aberrations? If so, how do you perform karyotyping? How do you test chromosomal aberrations?
Yes, karyotyping has been performed on most of our iPS cell lines. Currently, we do not provide karyotyping services. Our reliable partners conducted chromosome related assays.
This is regarding your Neural Stem Cell Line, Human: #hNSC11
Are these derived from Adult or fetal sources?
They are from adult sources.
From which tissue were these cell lines derived?
The cell lines are derived from human fibroblasts.
Were episomal vectors used to generate the iPSCs?
Yes. We used our episomal iPSC reprogramming kit (Catalog no. RF202).
Which protocol was followed to differentiate the iPSCs to NSCs?
We followed the Nature Protocols (ref?).
SV40 T Antigen Cell Immortalization Kit: catalog #CILV01
Is the virus replication-incompetent? If so, how do you recommend we confirm that there is no virus present in the cell cultures?
Yes. Any viral titering kit could detect the virus presentation. CONTACT US for more details.
Does the SV40 T Antigen get integrated into the host genome? What else gets integrated along with it?
Yes, SV40 T antigen does, as well as the puromycin resistance gene.
Aside from going out to 30+ passages to confirm the cells are immortalized, how can we confirm that the procedure was successful?
Based on our experience, it would be hard to confirm that.
We are using fibroblasts. How much puromycin is recommended for selection in fibroblasts?
We recommend that the final concentration of puromycin will be 0.5-1 ug/ml.
We are part of a government contractor doing work for NIH investigators. Does this qualify for the non-profit price for this product?
How do you generate iPSCs? How do you select and confirm the cells? Which cell types and what species have you successfully worked on?
IPSCs could be generated by our episomal or retroviral reprogramming methods, depending on the customer’s request. Please check our website or CONTACT US for more details. IPSC-like colonies will be picked by morphology and will be confirmed
by immunostaining of multiple pluripotency markers (e.g., OCT4). So far, iPSC was successfully generated from PBMC, fibroblast, dental pulp stem cell, etc., with species of human and mouse.
For your cell immortalization services, which method do you use? What are the differences between different methods? Pros and Cons?
We usually generate an immortalized cell line by overexpressed gene SV40 T antigen or hTERT. For more details, please CONTACT US. ALSTEM scientists are happy to provide a free consultation.
Virus packaging services: What are the differences between the 2nd and 3rd generation?
Briefly, the 2nd generation uses two plasmids system, while the 3rd generation uses three plasmids system, which is safer. Please check our website for more information.
For your transfection reagent, which cell types have you tried? What are the transfection conditions?
It works well in HEK 293 cells and would be efficient for other cell lines. Transfection conditions should be optimized for each cell type.
How to determine the sex of the anima from which the cells were derived, specifically, MEF-SV40 cells?
You may use the following PCR method to determine the gender of the MEFs.
In order to confirm sex in MEFs, cell lysate was used for PCR analysis. The Sry gene was amplified using forward primer 5’–TTG TCT AGA GAG CAT GGA GGG CCA TGT CAA—3’ and reverse primer 5’–CCA CTC CTC TGT GAC ACT TTA GCC CTC CGA– 3’.
Amplification ran as follows: 3 minutes at 94°C, a 35x repeated cycle of 30 seconds at 94°C/ 30 seconds at 58°C/ 40 seconds at 72°C, followed by 2 minutes at 72°C and holding at 4°C. Samples were analyzed by agarose gel electrophoresis, and
sex was determined based on the patterns of the bands present. The Sry locus ran at 273 bp. (Adapted from Murdaugh LB, et., al. PloS one. 2018;13:e0194767)
Which strain of mice iPS02M is derived from?
This mouse iPS cell line is derived from C57Bl/6 mouse embryonic fibroblasts (MEFs).
How is iPSC generated?
To generate iPSCs, we transfected these cells with the episomal vectors contain five reprogramming factors (Oct4, Sox2, Lin28, Klf4, and L-Myc) using electroporation. We followed the protocol from Yamanaka’s paper as follows.
Okita K, Matsumara Y, Sato Y, et al. A more efficient method to generate integration free human iPS cells. Nature Methods 8, 409-412, 2011.
I have recently purchased your hTERT Cell Immortalization Kit and am preparing experiments following the protocol provided. Step 5 of the protocol states to subculture cells and add the appropriate amount of puromycin for
stable cell line generation. Do you have a recommended concentration of puromycin to add for optimal results?
The optimal concentration of puromycin varies among different types of cells, eg. 0.5-1 ug/ml for fibroblasts, 1-2 ug/ml for HEK293 cells, 10 ug/ml for CHO. If you are not sure about the concentration, you may test the kill curve for the
cells using different puromycin concentration from 0.1 – 10 ug/ml.