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I have a GFP construct that I use and usually get anywhere from 50-80% GFP positive cells using FACS analysis while I get only 30-40% using a GFP-NFAT fusion protein driven by the same promoter. Do you have any suggestions?

The Amaxa™ Nucleofection™ Conditions are optimised for each cell type, the solutions and programs are cell type dependent and not vector dependant. The expression of the fusion protein depends on the localization of the protein transcription,...

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...

I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection™. Do you have any recommendations?

For some adherent cell lines we strongly recommend using RPMI instead of DMEM medium. As described in culture conditions of the cell line optimization kit protocol as well as the NIH3T3 (DSMZ) protocol, using RPMI instead of DMEM as "transfer and...

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.

Can I use the Nucleofector™ Technology for RNAi applications? How do I start?

Yes. The Nucleofector™ Technology can be easily applied for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type specific Optimized Protocol for DNA vectors (cDNA, shRNA or miRNA expressing plasmids) or...

Does it matter if the PBS used for monocyte enrichment contains calcium and magnesium, if the cells should be used later in Nucleofection™ ?

The PBS should be calcium and magnesium free to prevent clumping of cells. We routinely use PBS without calcium and magnesium for example Lonza Cat. No. 17-516F

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I have a GFP construct that I use and usually get anywhere from 50-80% GFP positive cells using FACS analysis while I get only 30-40% using a GFP-NFAT fusion protein driven by the same promoter. Do you have any suggestions?

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

I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection™. Do you have any recommendations?

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

Can I use the Nucleofector™ Technology for RNAi applications? How do I start?

Does it matter if the PBS used for monocyte enrichment contains calcium and magnesium, if the cells should be used later in Nucleofection™ ?

Do you have a protocol for the Nucleofection™ of Plasmodium berghei?

Can I order any of the Nucleofector™ Kit components separately?

Do you have a protocol for the Nucleofection™ of Plasmodium yoelii?

How can I determine the correct centrifugation speed for my particular centrifuge and rotor?