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Is your pmaxGFP control vector supplied with the Nucleofector™ Kits suitable for stable expression?

No. This vector is only supplied in our Nucleofector™ Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is only...

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Why do you have different Optimized Protocols for the Nucleofection™ of unstimulated and stimulated Human T cells?

We found that the stimulation of T cells not only changes the cells' function but also significantly alters the requirements for successful Nucleofection™. That's why Lonza developed two Optimized Protocols for transfection giving different...

Do you have any recommended methods for detaching human monocytes from the plate for assaying or before Nucleofection™?

We have had good results by incubating the cells in ice cold PBS for 10 minutes and then rinsing the plates. Alternatively, the cells can be detached without a medium change by gently pipetting the cell suspension up and down. Our experience has also...

Is it possible to use frozen primary CD34+ hematopoietic stem cells for Nucleofection™?

Yes. For cryopreserved PBMCs or enriched CD34 populations, we recommend culturing the thawed cells 1-2 hours in culture medium before Nucleofection™. Any further enrichment procedure after thawing is not recommended. Viability of frozen nucleofected...

How do you recommend that we isolate splenic lymphocytes from mouse spleens for Nucleofection™? Is erythrocyte lysis required?

For the preparation of mouse spleen cells we recommend cutting the spleen once and passing the tissue through a 100µM cell strainer or steel mesh using a plunger. The cells are flushed into a petri dish containing PBS. In order to remove fat, cell...

What are the critical steps for successful Nucleofection™ of monocytic cell lines like THP-1, HL60 and U-937?

To achieve optimal results, we strongly recommend following the Optimized Protocols in regard to cell culture conditions (medium, splitting cycle, density before Nucleofection), DNA amount and purity. Please also be sure not to exceed 90xg when...

Do you recommend positive selection or depletion for the purification of Human monocytes for Nucleofection™?

We only recommend the depletion method. For Example, the RosetteSep™ Isolation Kit for human monocytes [Stem Cell Technologies, Cat. No 15028]. The advantage of depletion is that the monocytes are left untouched by antibodies during the process.

Do you recommend a particular supplier for siRNA duplexes for use in Nucleofection™ ?

For siRNA applications using the Nucleofector technology we recommend siRNA duplexes from Dharmacon, part of Thermo Fischer Scientific.

Can I use the protocol for MCF7 cells for other breast cancer cell lines?

No. Often times closely related cell types will require completely different conditions for Nucleofection™. Therefore, you may want to consider performing a full optimization by using the Cell Line Optimization Kit. Please click on the related link...

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