Do you have a non-animal-origin enzyme for cell dissociation?

Product FAQ

Answer

Yes, the TrypLE™ products contain rProtease, a non-animal, trypsin-like enzyme used for the dissociation of attachment dependent cell lines. TrypLE™ enzyme has demonstrated the ability to dissociate cells cultured both in serum-free and serum-supplemented systems. The catalog numbers for TrypLE™ Select are 12563-011 (100 mL) and 12563-029 (500 mL). Some example catalog numbers for TrypLE™ Express include 12604-013 without Phenol red (100 mL) and 12605-010 with Phenol red (100 mL). Both formulations are also available in additional sizes. All these products are animal origin-free.

Answer Id: E11903

Was this answer helpful?

Yes
No
Thank you for your response

Based on the sodium bicarbonate levels in the medium, what CO2 percentage is recommended?

Product FAQ

Answer

If the media formulation contains:
- NaHCO3 (g/L) <1.5, it needs CO2 at 4%
- NaHCO3 (g/L) 1.5-2.2, it needs CO2 at 5%
- NaHCO3 (g/L) 2.2-3.4, it needs CO2 at 7%
- NaHCO3 (g/L) >3.5, it needs CO2 at 10%

However, there are some exceptions. Gibco™ DMEM has always been made according to Dulbecco’s original published formulation, with 3.7 g/L sodium bicarbonate. Customers have been using this medium in CO2 incubators ranging from 5-10% CO2 for decades, usually with no trouble maintaining physiological pH; this also depends on the cell type. Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).

Answer Id: E11904

Was this answer helpful?

Yes
No
Thank you for your response

When do I use the direct or indirect isolation techniques?

Product FAQ

Answer

The indirect technique is chosen when the antigen targeted by the primary antibody is expressed in low density on the target cell surface. This is due to the fact that free antibodies will find their target antigen more easily than antibodies linked to the Dynabeads™ magnetic beads. Also when using the indirect technique, an excess of free antibody can be added to the system, allowing ample opportunity for monoclonals to find the target antigen. Finally, an indirect technique can be useful when a cocktail of monoclonal antibodies is used to deplete unwanted cells during negative isolation of a cell type. This is because antibodies against all unwanted cell types can be added at once to the starting cell population, provided the antibodies are from one species. The antibody-coated cells can then be targeted with secondary-coated Dynabeads™ magnetic beads. The direct technique is chosen when a limiting amount of monoclonal antibody is needed for targeting the cells of interest during positive isolation or depletion (e.g., when the target antigen is present at high density). It can also help when the possibility of interaction from the secondary antibody needs to be avoided, or if a stock preparation of primary coated Dynabeads™ magnetic beads is desired. Additionally the direct technique can be used when you do not want to cover all antigen sites with antibody (e.g., when you want to analyze the isolated cells by flow cytometry).

Answer Id: E4666

Was this answer helpful?

Yes
No
Thank you for your response

Can you provide some information about CD14 isolation?

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

When running the PrimeFlow™ RNA Assay in a 96-well plate format, I have spun the plate and cannot see my cell pellet. Is this normal?

Product FAQ

Answer

Depending on the starting cell number and the cell type used, the cell pellet may not be easily visible. For 2 million cells of PBMC, an opaque cell pellet is barely visible in a v-bottom plate.

Answer Id: E14489

Was this answer helpful?

Yes
No
Thank you for your response

What factors can contribute to rapid cell death/culture failure?

Product FAQ

Answer

There are a number of events that can contribute to this:

1. Incorrect CO2 levels-monitor the level of CO2 manually with a Fyrite kit, available from Bacharach (http://www.bacharach-inc.com/fyrite_analyzers.htm). Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO2 level. Check the settings to insure that CO2 levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
2. Temperature fluctuations in the incubator-Monitor the temperature of incubator with a good thermometer inside the incubator.
3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations-use at recommended levels.
4. Humidity is incorrect-check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO2 levels are largely irrelevant for most cultures if the humidity level is not high enough).
5. Incorrect osmotic pressure in medium-check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
6. Contamination by microorganisms-bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
7. Inappropriate medium is being used-double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

Answer Id: E11905

Was this answer helpful?

Yes
No
Thank you for your response

Can a cocktail of primary antibodies be added to a cell suspension in order to pull out several target cell populations simultaneously, using one secondary-coated Dynabeads™ magnetic beads product?

Product FAQ

Answer

Yes, a cocktail of primary antibodies can be added to a cell suspension in order to pull out several target cell populations with one secondary-coated Dynabeads™ magnetic beads product.
The Dynabeads™ magnetic beads Pan Mouse IgG (110.41; 110.42) works very well with a cocktail of mouse IgGs for the simultaneous capture of multiple cell types. it is recommended that you use an indirect technique with antibody cocktails (add all Ab to cells, wash off excess Ab, then add beads to capture Ab-coated cells).

Answer Id: E4667

Was this answer helpful?

Yes
No
Thank you for your response

I am running my Tris-Glycine gel using the XCell SureLock™ Mini Cell and the gel run is taking longer than usual. What could be causing this?

Product FAQ

Answer

Here are possible causes and solutions:

1) Buffers are too dilute: Check buffer recipe; remake if necessary.
2) Upper buffer chamber is leaking: Make sure the buffer core is firmly seated, the gaskets are in place and the gel tension lever is locked.
3) Voltage is set too low: Set correct volatage

Answer Id: E10608

Was this answer helpful?

Yes
No
Thank you for your response

How can macrophages be removed from samples?

Product FAQ

Answer

We have several methods to deplete macrophages and monocytes from a sample. First, we have an anti-CD14 coated bead that will specifically bind to macrophages, monocytes, and some granulocytes expressing the CD14 antigen. This bead will efficiently deplete such cells from a variety of starting samples such as whole blood, buffy coat, peripheral bloods mononuclear cells, single-cell suspensions from tissue, etc. with greater than 98% depletion normally achieved.

It is also possible to exploit the phagocytic capabilities of these cells by adding virtually any bead, which will be internalized by these cells. After a relatively short period of time, the cells with engulfed beads can easily be removed on a magnet.

All phagocytic cells will phagocytose Dynabeads™ magnetic beads given the opportunity, and will then be co-isolated with the target cells when the sample is put on a magnet. If you have any problems with this, try adding beads that have been deactivated (e.g. boiled to damage the antibodies) to the sample (to feed them to the macrophages) and incubate for 45 min, after which the phagocytic cells will have phagocytosed the beads and can be removed using the magnet. After removing these cells, proceed as usual with room temperature incubations.

Answer Id: E6120

Was this answer helpful?

Yes
No
Thank you for your response

When running the PrimeFlow™ RNA Assay, will the use of 96-well plates increase the amount of cell loss compared to the tube protocol? What type of cell loss should I expect?

Product FAQ

Answer

The cell loss may be slightly higher in 96-well plates compared to the tube, depending on the method used to remove supernatant after washes (manually or using an aspirator), but should be less than 50%.

Answer Id: E14490

Was this answer helpful?

Yes
No
Thank you for your response

I want to reseal plates after I resuspend the siRNAs. What do you recommend?

Product FAQ

Answer

For those researchers who want to re-seal plates after resuspension, we recommend aluminum seals over plastic. We have used the following seal in the past with good success:

Catalog Number: 47734-816
Supplier: Axygen Scientific
Description: Aluminum Sealing Film. Maintains excellent seal on microplates under the widest temperature conditions (-80 degrees C to 97 degrees C) and reagents.

Answer Id: E6576

Was this answer helpful?

Yes
No
Thank you for your response

Can I autoclave agarose to prepare agarose gels?

Product FAQ

Answer

No. Autoclaving at temperatures that are too high or pressures that are too high can hydrolyze the agarose polymer and weaken it.

Answer Id: E2921

Was this answer helpful?

Yes
No
Thank you for your response

How do I isolate RNA from liver or other organs?

Product FAQ

Answer

Isolating high-quality RNA is critical to the overall success of in vivo experiments.

Answer Id: E9814

Was this answer helpful?

Yes
No
Thank you for your response

Can I use the PureLink™ Quick Gel Extraction Kit (Cat. No. K210012) to purify supercoiled DNA?

Product FAQ

Answer

Supercoiled DNA will not be eluted from the membrane using the standard Wash Buffer that contains 70% ethanol. Supercoiled DNA can be eluted from the membrane only when the Wash Buffer contains 50-55% ethanol.

Answer Id: E5069

Was this answer helpful?

Yes
No
Thank you for your response

Can you use sealed culture vessels instead of vented ones in incubator instead of humidifying when culturing insect cells?

Product FAQ

Answer

No. Suspension cultures will quickly become oxygen limited and will only reach cell densities of 1 to 1.5 x 10E6 cells/mL. Recombinant product expression will also be greatly reduced. We recommend using plastic Erlenmeyer shake flasks with screw cap tops (not foam plugs). This allows you to cover and keep the cultures sterile and allow free gas exchange.

Answer Id: E11906

Was this answer helpful?

Yes
No
Thank you for your response