Product FAQ

After I labeled neurons with MitoTracker™ Red CMXRos, they are dead the next day. Is this expected?

Answer

The Mitotracker™ dyes should be imaged soon after staining because over time, those dyes can be toxic.

Answer Id: E14896

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Product FAQ

I keep getting low transduction efficiency when using CellLight™ labeling reagents on my neurons. What can I do to improve the efficiency?

Answer

Neurons are more difficult to transduce than many other cells. The main way to improve transduction is to label with a higher number of particles per cell. For primary neurons, it can also help to transduce them at the time of plating rather than on established cultures. There can also be a slower onset of expression in neurons and peak expression often occurs on day 2-3 rather than 16 hours after transduction.

Answer Id: E14897

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Product FAQ

I labeled my neurons with DiI and then fixed and permeabilized and now I have no signal. What did I do wrong?

Answer

DiI is a lipophilic dye that resides mostly in lipids in the cell, when cells are permeabilized with detergent or fixed using alcohol this strips away the lipid and the dye. If permeabilization is required CM-DiI can be used because this binds covalently to proteins in the membrane; some signal is lost upon fixation/permeabilization, but enough signal should be retained to make detection possible.

Answer Id: E14898

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Product FAQ

Am I required to use fresh PCR product with the BigDye™ Direct Cycle Sequencing Kit, or can I store the PCR reaction before proceeding? What temperature is best for storage?

Answer

We always recommended that you use fresh PCR product, but if you need to store it, the amplified PCR reaction can be stored for 16 hours at 4°C or up to 4 weeks at –20°C.

Answer Id: E6300

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Product FAQ

I stained my cells with a lipophilic cyanine dye, like DiI, but the signal was lost when I tried to follow up with antibody labeling. Why?

Answer

Since these dyes insert into lipid membranes, any disruption of the membranes leads to loss of the dye. This includes permeabilization with detergents like Triton™ X-100 or organic solvents like methanol. Permeabilization is necessary for intracellular antibody labeling, leading to loss of the dye. Instead, a reactive dye such as CFDA SE should be used to allow for covalent attachment to cellular components, thus providing for better retention upon fixation and permeabilization.

Answer Id: E14899

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Product FAQ

When using the Live/Dead™ BacLight™ Bacterial Viability and Counting Kit, for flow cytometry, some cells seem to have both red and green signal. Are these cells dead or alive or dying?

Answer

The green dye in the kit will label all the cells as it is a cell-permeant nucleic acid stain. The red dye is not cell permeant, and will only stain the cells with compromised membranes (dead cells). Therefore, any cells with red signal will be considered dead. It is possible that you will have some cells that are only red, some that are red and green, and some that are only green. Sometimes the red dye will displace the green dye. In any case, any red cells are dead.

Also, the green dye emission may bleed through into the red channel. Do a single-color staining and examine under both green and red filter sets to determine the level of bleedthrough. To avoid this bleedthrough, use a lower concentration of dye, and, if possible, use narrow bandpass filters.

Answer Id: E14916

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Product FAQ

With the Single Cell-to-CT™ Kit, are there any special considerations when evaluating expression of GC rich genes?

Answer

There are no special considerations needed for GC rich genes.

Answer Id: E7099

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Product FAQ

Do you have any recommendations for the pre-PCR template DNA concentration in the BigDye™ Direct Cycle Sequencing Kit procedure?

Answer

The recommended DNA input amount is 4 ng.

Answer Id: E6301

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Product FAQ

What is the total number of probes in OncoScan™ FFPE Assay Kit?

Answer

OncoScan FFPE Assay Kit has 217,454 probes.

Answer Id: E13849

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Product FAQ

I used one of your FluoroMyelin™ stains and noticed it stains cells other than glial cells. Is there something wrong with the product?

Answer

FluoroMyelin™ is a lipid stain, any lipid can be stained by it but there is a higher lipid content in myelin that it will stain much more intensely than other membranes.

Answer Id: E14900

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Product FAQ

I dried my gel using Gel-Drying Solution and it dried milky white. Can the gel be recovered?

Answer

Gels turn white when they are dried too rapidly, usually in an environment that is too ventilated, drafty, or warm. You can remedy this problem by dabbing the gel with deionized water on a Kimwipe™ tissue to rehydrate the white areas. Re-dry the gel in a dark, dry, non-drafty place, such as a drawer or cabinet.

Answer Id: E11037

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Product FAQ

Which controls are included in the new GeneChip™ Exon array?

Answer

A variety of controls are included on the Exon Array, designed specifically to facilitate the application of genome-wide exon-level expression profiling. These controls include:
Intron controls--for approximately 100 genes with relatively constitutive expression, both exon-based and intron-based probe sets were tiled. The intron/exon normalization control probe sets can be used to monitor contamination from genomic DNA, hnRNA, as well as to provide a baseline for experiment quality control.
Hybridization controls--bioB, vbioC, bioD and cre
Poly-A RNA controls--lys, dap, phe, and thr

Answer Id: E13550

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Product FAQ

How can I remove mycoplasma contamination from my cell culture medium?

Answer

Very often, mycoplasma contamination cannot be removed from the culture so it should be discarded. You may have a unique culture that you prefer not to discard and would like to try to clean it. Ciprofloxacin and Plasmocin™ have reportedly been used for this application. If interested in a protocol or directions for use, check with the antibiotic supplier or published literature. Note that mycoplasma are very difficult to remove from culture and spread easily so the treated cultures should be quarantined until clear of mycoplasma and the laboratory should be thoroughly cleaned.

Answer Id: E14917

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Product FAQ

Can you provide some information about CD14 isolation?

Answer

Some important considerations for CD14 cell isolation include:

(1) Beads per volume of sample is more important than how many beads are needed per target cell for efficient isolation (the optimum is 4 but 1 - 3 is probably enough if you have a small volume). This is a function of concentration and binding kinetics. In order for the beads to reach the target, they needs to be distributed uniformly in the solution, since they are probably held stationary in the solution. The minimum concentration to use is 2 x 10e7 beads per mL, independent of the number of target cells.

(2) There is a correlation between the amount of bound antibody coupled to the beads and the ability of the beads to be attracted to the magnet. However, you will reach a threshold of antibody:bead coupling. The CD14 bead has not been optimized to handle the presence of soluble CD14. Empirically we have seen that one wash is required to attract bound CD14 cells to the magnet.

(3) Phagocytic activity must be considered. The phagocytic activity will be slowed as the temperature drops. This is also true for endocytosis. As the beads are too large to enter via endocytosis, this is not a problem. However, the dedicated "eaters" like monocytes have the capacity to engulf larger materials like Dynabeads™ magnetic beads. A way of inhibiting this is to reduce the temperature to a point where phagocytosis is blocked.

(4) The efficiency of using Dynabeads™ magnetic beads to “pull” bound cells toward the magnet is correlated with the amount of antibody coupled to the beads (up to a plateau).

(5) The binding properties of the CD14 beads also must be considered because they are two-layer beads. The functional amount of CD14 antibody on the bead is less than 0.5 μg per 10e7 beads. This is due to the production method: 1 μg CD14 antibody is coupled onto HAM6-beads, which have 1 μg HAM6 coupled. The capacity of the HAM6 bead for IgG2a is less than 0.5 μg per 10e7 beads. Thus, using 2 x 10e7 beads per mL of sample will give a maximum binding capacity of 2 x 2 x 0.5 μg CD14-antigen = 2 μg CD14 (assuming that both arms of the CD14 antibody can bind CD14 antigen, which may be an overestimate due to sterical hindering).

Answer Id: E6119

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Product FAQ

Have the Cells-to-CT™ kits been used with plant tissue?

Answer

We have not tested plant tissue internally. When working with plants, we suggest employing one of the following standard homogenization methods: (1) motorized homogenizer, (2) stainless steel bead beating, or (3) liquid nitrogen-mortar/pestle.

Answer Id: E7100

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