达科为生物技术有限公司 -- 常见问题

A. There are several reasons to choose the Amaxa® Nucleofector? Technology for your gene transfer experiments. One is the direct transfer of DNA into 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.

A. In most cases none. For each cell type in our product list, we offer a cell-type-specific solution and thoroughly optimized electrical parameters. These are already pre-programmed in the Nucleofector® Device. Our Amaxa? Optimized Protocols give you recommendations regarding cell culture and handling for optimum transfection results.

A. Lonza is constantly developing new Amaxa® Nucleofector? Kits and Optimized Protocols for an increasing number of primary cells and cell lines. In order to obtain the latest information, check our website or contact our Scientific Support Team. If your target cell is a cell line, we offer the Cell Line Optimization Nucleofector? Kit. This kit enables a rapid and simple determination of the optimal Nucleofection? Conditions for your cell line of interest, including those which are difficult-to-transfect.

A. The recommended cell number will vary depending on which Amaxa? Optimized Protocol is being used. In general for the Nucleofector? Device (single cuvette format), using less than 2x105 cells per reaction causes a major increase in cell mortality. For some cell lines, we have tried cell numbers up to 3x107. The problem here is that you end up with a very large cell pellet leading to a dilution of the Nucleofector? Solution as the maximum cuvette volume is 100 μl to 110 μl. We suggest increasing the cell number successively from experiment to experiment but not exceeding a cell pellet volume of 50 μl. Our Small Cell Number Nucleofector? Kit for primary neurons represents an exception to this. It comes with different cuvettes designed for 20 μl sample volume. With the Small Cell Number Nucleofector? Kit 20,000-100,000 cells per sample are used. For the 96-well Shuttle? System, the sample volume is 20 μl (typically cutting the cell number recommended for the single cuvette by 1/5). The lower limits are often dictated by the minimum amount of cells required for optimal plating density on 96-well cell culture plates.

A. 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 passages.

A. We routinely use plasmids of 4-7 kb in our laboratories and plasmids up to approximately 20 kb should not be a problem. The optimized conditions are cell-type-specific. That means you can use the identical set up for DNA, siRNA, mRNA,

peptide and large molecules like bacterial artificial chromosomes (BACs) as well. It is very likely that you will see a decrease in the transient efficiency when BACs are used as a substrate, but in general it works very well. You also have to consider that a) by using 5-10 μg DNA you have less copies in your experiment than with standard constructs which leads to lower brightness of the cells, and b) the quality of the DNA is very important. So try to use endotoxin free DNA preparations, and check the DNA quality on an agarose gel to be sure that the DNA isn’t nicked.

1) ToxiLight? Non-Destructive Cytotoxicity BioAssay Kit. ToxiLight? is a patented luminescent assay that measures the release of adenylate kinase. Unequivocal results are available in less than 10 minutes. It can detect as few as 10 dead cells per well without requiring a lysis step. Please see more details on page 226.

2) Flow cytometry determination of viable/dead cells by propidium iodide staining. We normally analyze transfection efficiency in living cells by flow cytometry: 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 flow cytometry analysis. This helps ensure that our data is complete and accurate.

3) Flow cytometry 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-labeled beads from BD Biosciences (to detect APC your flow cytometry 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 flow cytometry. To count the beads by flow cytometry 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):

A. In principle, co-transfection of different plasmids should not be a problem. However, the total amount of DNA used is a crucial point because high amounts of DNA could cause decreased cell viability. For the Nucleofector? Device (single cuvette format), we recommend starting co-transfection experiments with 1 μg - 5 μg total DNA first and then successively increasing the DNA amount if necessary. Also verify that both plasmids will be expressed in this cell type. For the 96-well Shuttle? System you should calculate 1/5 of the amount; 0.2 μg - 1 μg DNA per vector.

A. Yes. The Amaxa? 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).