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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...

When nucleofecting cancer cell lines, do I need to be concerned about passage number?

For the most efficient gene transfer, we recommend using cells that are in logarithmic growth phase and at a passage number of 10-15 (from the time of thaw). This is because some cell lines differentiate and change their features after many...

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...

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.

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. BEBP17-516Q

Is the age of the mice important for my mouse T cell Nucleofection®?

Yes. We recommend using mice between 6-12 weeks. Using mouse T cells isolated from younger or older animals for Nucleofection® may result in much lower transfection efficiencies and/or viabilities.

I see activation of my monocytes (or macrophages or DCs) following Nucleofection®. Why is this? What can I do to address this problem?

We have examined the effects of Nucleofection® (without DNA) on these cells and have not observed significant activation. This indicates that neither the Nucleofector® Solution, the Nucleofector® Program nor the recovery medium are...

Why am I not able to detect my fluorescent labeled siRNA (e.g. FITC) after 24 or 48 hours post-Nucleofection?

Fluorescently labeled siRNA duplexes can be used to analyze transfection efficiency by fluorescence microscopy or flow cytometry. However, FITC, Rhodamine, or Alexa-488 labeled siRNA oligos should be analyzed 0.5-3 hours post-Nucleofection™. Cy-5...

Is there a difference between, for example, the programs C-20 and C-020?

No, the programs C-20 and C-020 are identical regarding their electrical parameters. The letter minus a two-digit number is the nomenclature of the Nucleofector™ I. Since we have launched the Nucleofector™ II we changed it to a letter minus a three...

Why do you recommend 7AAD instead of PI staining for cell determination in mouse macrophages?

The results obtained using 7AAD staining are much clearer than those obtained using propidium iodide (PI). Using flow cytrometry stained and unstained, transfected and non-transfected populations can be separated much more easily.

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