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What method can I use to fix my HL-60 cells as shown in your optimized protocol?

The cells which are shown in the HL-60 optimized protocol are living cells and not fixed cells. However a good method for fixation of GFP is to use 3.5%-4% Paraformaldehyde in PBS. Please keep in mind that the fixation method is dependant on what you...

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...

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...

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. 17-516F

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

The correct rotor speed can be calculated by measuring the maximum radius of your rotor and entering the information into the table found on the website which can be reached by clicking on the Related Link.

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.

Does the recovery medium contained in the Mouse T Cell Nucleofector Kits contain any immunogenic substances?

No. The post Nucleofection™ Recovery media contains no immunogenic factors and should not influence cell stimulation or differentiation. For Mouse T cells, some experiments have shown that after Nucleofection™ cells can be stimulated efficiently in...

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 sufficient for...
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