2020-12-11 11:31:40 youzre.com 185

Q:   What is the basic principle of the Nucleofector Technology?

A:   The Nucleofector Technology is a novel transfection method especially designed for the needs of primary cells and difficult-to-transfect celllines.

It is a non-viral method based on a unique combination of electrical parameters and cell-type specific solutions.With the Nucleofector Technology the DNA is transported directly into the nucleus. Also with other substrates, such as siRNA or miRNA, Nucleofector Technology offers highly efficient and robust transfection with high cell viability.

Q:   Why is the Nucleofector Technology ideal for primary cells and difficult-to-transfect cell lines?

A:   There are several reasons to choose the Nucleofector Technology for your gene transfer experiments. One is the direct transfer of DNA to the cell nucleus. This makes gene expression independent from cell division. Therefore, the technique is the ideal tool for primary cells, even for non-dividing cells such as neurons. In cell lines as well as primary cells, this yields gene expression shortly after transfection and in many cases transfection efficiencies of over 50% can be detected in as little as 2-4 hours.

Q:   What is the best way to establish the Nucleofector Technology in my lab?

A:   We strongly recommend establishing the Nucleofector Technology with the positive control vector pmaxGFP provided in our kits. pmaxGFP encodes the green fluorescent protein (GFP) from Copepod Pontellina sp. Just like eGFP expressing cells, maxGFP expressing cells can easily be analyzed by fluorescence microscopy or flow cytometry to monitor transfection efficiency.

Q:   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?

A:   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 recover" medium can be crucial for the survival of the cells whether you work with adherent or suspension cells. For some cell types, we see a very high mortality if we use DMEM immediately after Nucleofection. Immediately after Nucleofecting your cells, we recommend that you add 500 µL's of pre-warmed RPMI+ 10% FCS to the cuvette and then remove the entire contents of the cuvette and place it into an Eppendorf tube at 37 C° for 5 -10 minutes to allow the cells to recover. After that time you can plate them in the appropriate media. There is no need to get rid of the RPMI, just add the cell suspension to the DMEM containing well. It is also possible to change the media after 5-6 hours.This will lower the mortality especially with 293 cells.

Q:   How can I increase cell viability of Dendritic cells after Nucleofection?

A:   DNA amount and quality are very critical for Nucleofection of Dendritic Cells (recommended 0.5-1µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic cell culture and transfection, e.g. PBS, FCS and especially DNA, are Endotoxin free. We additionally recommend purifying your DNA by precipitating it twice with 20% PEG/2.5 M NaCl. Viability is usually not an issue when working with mRNA or siRNA.

Q:    My Nucleofector Solution was frozen. Is this a problem?

A:    As long as the supplement was not added to the Nucleofector Solution, then there is no risk of any damage to the solutions. Even long-term storage of several months did not alter the performance of the Nucleofector Solution. However, if the supplement was added and then frozen, check to see if there is any precipitate. If there is not, then the solution should be fine. If precipitate is present, it may be worthwhile to do a test Nucleofection Reaction to make sure the solution is working properly.

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

A:   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 oligonucleotides (siRNA, miRNA inhibtors). Prior to beginning comprehensive experiments multiple parameters associated with experimental design need to be optimized: Selection of appropriate controls (non-targeting siRNA, siRNA targeting a house-keeping gene, untreated cells). Determination of minimum effective siRNA amount for your target gene in your cell type of interest. Determination of the optimal analysis time point (kinetics of down-regulation depend on target gene, cell type and analysis level). Identification of a suitable and robust analysis method (i.e. mRNA, protein or phenotypic analysis). Please click on the associated link for example data and recommendations.

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

A:   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.

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

A:   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 the recovery medium containing FCS.

Q:   Can I order any of the Nucleofector Kit components separately?

A:   No. The Nucleofector Kits are only available as full kits.

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

A: 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 cell activation. Please note that the recovery medium used for these experiments did not include serum or any other supplements.

There are several possibilities that could account for the activation you have observed:

1. Leukocytes are very sensitive to LPS (human leukocytes much moreso than those from mouse) so it is very important for the DNA to be as pure as possible - above our normal recommendations. Namely, that there are two additional PEG precipitations to clear out residual LPS from the DNA prep on top of the endotoxin-free purification with the 1.8 OD ratio. These extra purification steps are important, since even trace amounts of LPS are sufficient to activate antigen presenting cells (APCs) like DCs, monocytes and macrophages.

2. Plasmid DNA alone can induce activation. This is a natural response of APCs to the presence of unmethylated CpG dinucleotides (i.e. characteristic of bacterial and viral DNA). So even removing all of the LPS from DNA preparations will probably not eliminate activation. A reference for this is Gene Ther. 1999 May;6(5):893-903 3) We tested only unsupplemented recovery medium (i.e. without added serum). If you have added anything to the recovery medium it will be important to verify that none of these components can induce activation. According to our R&D the cells seem fine for up to 48 hours culture without serum. However if you subsequently wish to stimulate the cells you may require serum in the medium.

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

A: 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 electrical parameters for stimulated and unstimulated T cells. In addition, the protocol is optimised for 5-6d stimulated T cells. If you use allostimulated or long term stimulated T cells like LAK cells, please contact our Scientific Support Team for assistance.

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

Q:   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 cells might be lower than that of fresh ones.

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

A:    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 labelled siRNAs can be detected up to 24 hours post Nucleofection. 

Likewise, if one is attempting to visualize a label via microscopy, you may have to use a concentration that is much greater (5µg-10µg per sample) than that required for functional analysis. Some labels are also subject to photo bleaching, pH changes, and may exhibit cell type specific effects as well. 

To minimize FITC-bleaching it may help to wrap the culture plates in aluminum foil immediately after Nucleofection. So, it is not possible to both determine efficiency and look for functional knockdown in the same experiment. A better and more cost effective positive control is to use a known sequence to knock down an endogenous housekeeping gene.

Q:   What is a Neural Stem Cell (NSC)?

A:   Neural stem cells are adult stem cells but you also find them in embryos (already in "adult" differentiation status) and they have the potential to develop into neurons and glial cells.

We recommend isolating them from rat and mouse embryos because more cells can be obtained from embryos than from adults. For Nucleofection of NSC from foetal or adult brains, you can use the same Optimized Protocol. Neuronal progenitor cells already have a neuronal phenotype but are not mature neurons yet. They have a lower differentiation potential than neural stem cells.

Q:   Does your Nucleofector Solutions contain anything that would inhibit attachment of adherent cells?

A:   No.In many cases where decreased attachment is a problem, the cause is inactivated trypsin. Unless the trypsin is inactivated with trypsin inhibitor or media containg BSA or serum, it will continue to degrade the cells and ultimately decrease cell viability. As a general rule, as soon as the cell monolayer begins to detach, add media or trypsin inhibitor to the culture dish. Alternatively, one can use a cell scraper to detach the cells.

Q:   After Nucleofection, how do you determine cell viability?

A:   We determine cell viability after Nucleofection in two ways:

1) FACS determination of viable/dead cells by PROPIDIUM IODIDE STAINING. We normally analyze transfection efficiency in living cells by FACS: We first exclude cellular debris by gating for the "normal" population (regarding size and granularity) in the forward-sidescatter. From this gated population we determine dying cells by propidium iodide staining and exclude them from analysis by setting another gate. So, only those cells which are in the FSC-SSC gate and are not propidium iodide positive are analyzed for efficiency. To be even more precise in the determination of the mortality for adherent cells, we also collect the detached cells in the supernatant and combine these after trypsinization with the former adherent cells and include them in the FACS analysis. This helps ensure that our data is complete and accurate.

2) ViaLight Plus BioAssay Kit: We determine the amount of live cells after Nucleofection using our ViaLight Plus Cell Proliferation and Cytotoxicity BioAssay Kit

3) FACS determination of total cell loss induced by Nucleofection (optional method): In order to get an idea about the total cell loss, we compare the initial cell number per sample with the final cell number per sample. For that we use APC-labelled beads from BD Biosciences (to detect APC your FACS needs a 4 channel laser, because APC is detected on channel

4). We prepare a stock of beads (1000 beads/µl in PBS). After 5 minutes of vortexing, we add 50 µl of the beads to the sample we want to analyze by FACS. To count the beads by FACS you have to lower the threshold in the FSC/SSC down to 20 because they are much smaller than cells. After analysis, we do the following calculation to define the number of used living cells in the analyzed sample (input X, unknown value): input X = number of used beads*/counted beads x counted cells in R2** * input: 50µl = 50000 beads **R2 gate means: normal cell population in FSC/SSC without cellular debris (gate R1) AND without PI-postive cells in FL-2/FL-3. By comparing the X-values for the untreated sample and the nucleofected samples you can calculate how many dead cells you have in your treated samples.

Q:   Can I use larger cuvettes for my Nucleofection Reaction ? Can I use the cuvettes more than once?

A:   No.The electrical parameters provided by the Nucleofector Device are optimized for the cuvettes contained in the Nucleofector Kits.

The cuvettes are single-use only. Using the cuvettes more than once will result in higher cell death and lower transfection efficiency. Furthermore, cross-contamination e.g. with residual DNA may occur, since Nucleofection is a very efficient delivery method.