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Please compare your cells-only plate to the infected plate. The uninfected cells should appear overgrown when compared to infected cells, as transfection inhibits growth. If this is seen, keep checking the infected cells daily for other signs of infection (nuclear swelling, detachment from the plate, viral budding, and lysis). The kinetics of infection may be slower than expected. If cell growth does not appear to be inhibited, you may consider the following factors:

- How was the DNA prepared? We recommend using a resin-based DNA purification system, such as our PureLink HiPure Plasmid Prep Kits.
- Were cells in log phase? What was their confluency? We recommend that cells be in log phase, 95% viable, and plated at 50-70% confluency for successful transfection.
- What transfection reagent was used? We recommend using Cellfectin II Reagent.


Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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Luminex xMAP technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For ProcartaPlex multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex instruments for analysis.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

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ProcartaPlex multiplex assays, which are based on Luminex xMAP technology, provide a versatile platform that gives users more flexibility and a greater array of options for analyte detection. Whether you are testing for single or multiple analytes, ProcartaPlex multiplex assays deliver accurate analytical performance using efficient, easy-to-follow protocols. Each of these assays has undergone the same development, validation, manufacturing, and quality control standardization we conduct for our ELISAs. Each lot of ProcartaPlex multiplex assays as well as ELISA assays is fully qualified with the appropriate sample type (i.e., species-specific serum, plasma, and cell culture supernatants), and each lot is evaluated based on the following performance characteristics:

Specificity-each analyte is screened to make sure there is no significant cross-reactivity with other analytes in the multiplex test
Sensitivity-each analyte is evaluated for both functional sensitivity (differentiation from background) and lower limit of detection (LLOD)
Precision/accuracy-multiplex assays have good intra-assay precision (<10% CV), inter-assay precision (<10% CV), and lot-to-lot consistency (<20% CV); these values are comparable to or better than most ELISA tests
ProcartaPlex multiplex assays are regularly tested against the matching ELISAs. Therefore, you can switch easily from ProcartaPlex assays to ELISA and vice versa with reliable results. Most of our ProcartaPlex assays use the same antibody pairs as our traditional plate-based ELISAs, resulting in high correlation (R2 > 0.9) between the two assays.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

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The PSC cryopreservation kit contains xeno-free PSC Cryopreservation Medium, which is a ready-to-use solution for the cryopreservation of early passage pluripotent stem cells (PSCs), and Gibco Revitacell Supplement (100X), a chemically defined recovery supplement for use in the post-thaw culture medium. When used in combination, these reagents help minimize loss of cell viability, maximize post-thaw recovery, and minimize unwanted differentiation of PSCs. This kit can also be used to cryopreserve and recover peripheral blood mononuclear cells (PBMCs) to improve post-thaw cell viability and recovery.

Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.

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There are several options. Our first recommendation is to use the Invitrogen MycoFluor Mycoplasma Detection Kit.

Mycoplasmas are small, self-replicating prokaryotes (0.3 - 0.8 mm diameter), that lack a cell wall and have the ability to adsorb onto host cells. Mycoplasma is one of the most serious forms of cryptic contamination and its presence is not detected unless appropriate tests are made or until some aspect of cell behavior is noticed to have changed. Between 15 and 50% of cell lines submitted to cell banks are contaminated with mycoplasma. Mycoplasma spreads readily among cell lines via reagents and media, the operator and the work surface.

The presence of mycoplasma may invalidate the results obtained with that culture. The presence of mycoplasma-infected cultures can result in the shut-down of the entire laboratory until the infection can be eliminated, whereupon complete restocking is required. The origin of contamination is usually traced to mycoplasma present in animal (bovine) serum or to human oral mycoplasma transferred by droplet infection during cell culture. The simplest test for the detection of mycoplasma in cultures is the use of a fluorescent dye which binds directly to DNA causing fluorescence (e.g. Hoechst 33258) which can be seen by fluorescence microscopy. Mycoplasma positive cells will show intense fluorescent spots on the plasma membranes or show filaments which may be absorbed onto the cells. Uncontaminated cells show only brightly fluorescent cell nuclei. The technique is rapid (less than 30 minutes), but requires heavy contamination (10E6 mycoplasma/ml) to produce a clear positive result. If however, the suspect cells are co-incubated for 2-4 days with an "indicator" cell line (such as 3T3) which is particularly suitable for demonstration of positive staining, then sensitivity can be substantially increased. Microbiological culture techniques are available that operate at a greater sensitivity, but it can take up to 21 days to obtain a result, a positive control is needed, and the result may require expert interpretation.

A variety of PCR-based methods are available, some of which have been utilized as commercially available detection kits. It is recommended to use a combination of DNA staining and a PCR-based method once every 3 months for all growing cultures in the laboratory and for every new cell line as it enters the laboratory. In addition, all Master and Working Cell Banks should be tested at the time of freezing. Quality control and good working practice will reduce potential problems. It is important that frozen stocks are created immediately after testing and re-tested before distribution. If cells are cultured for more than 3 months after testing, they should be re-tested. Regulatory bodies now insist that cell cultures used for the production of reagents for diagnostic kits or therapeutic agents are free from mycoplasma infection. Also, some scientific journals have the policy of requiring statements from authors that the culture work reported in those journals is carried out with mycoplasma-free cells. Normally, when contamination with mycoplasma is apparent, the recommendation would be to discard the cultures and start again. If necessary, and only if the contamination is not extensive, then it is often possible to rescue the cells by treatment with one of the commercially available antibiotics. This must only be considered for a remedial action, not as a routine supplement to growth media (and thereby a substitute for good cell culture practice).

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Below are possible reasons why you may see reduced viability following transfection, along with suggested solution.

1. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µl) ratio of 1:2to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine 2000 (µl) ratios from 1:0.5 to 1:5.
2. Possible Cause: Plasmid DNA preparation contains high levels of endotoxin
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPureNucleic Acid Purification Kits.
3. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
4. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
5. Possible Cause: Complexes not thoroughly mixed in growth medium
   Suggested Solution: Following addition of transfection complexes into medium, ensure that the plate or wells are thoroughly mixed to prevent concentration of DNA:transfection reagent complexes in the wells
6. Possible Cause: Cells have changed over time, or splitting conditions have changed
   Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
7. Possible Cause: Antibacterial agents were used in growth medium during transfection
   Suggested Solution: Do not use antibiotics such as chloroquine, penicillin, or streptomycin in growth medium because during transfection, cells are more permeable to antibiotics, which may cause toxicity.
8. Possible Cause: Transfection reagent stored improperly
   Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.
9. Possible Cause: Cationic lipid reagent was oxidized
   Suggested Solution: Do not vortex or agitate cationic lipid reagents excessively; this may form cationic lipid reagent peroxides.
10. Possible Cause: Selection antibiotic added too soon
    Suggested Solution: When creating stable cell lines, allow at least 72 hr for cells to express the resistance gene before adding selective antibiotic.

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Below are possible reasons why you may be getting low transfection efficiency, along with suggested solutions:

1. Possible Cause: Plasmid DNA, siRNA, or transfection reagent diluted in media containing serum or complexes formed in the presence of serum
   Suggested Solution: Use serum-free medium for dilutions of plasmid DNA, siRNA, and transfection reagents. Note: we recommend using Opti-MEM | Reduced Serum Medium (Cat. No. 31985-062)to dilute Lipofectamine 2000 and DNA before complexing.
2. Possible Cause: DNA: transfection reagent ratio sub-optimal for cell line
   Suggested Solution: Prepare complexes using a DNA (µg) to Lipofectamine 2000 (µL) ratio of 1:2 to 1:3 for most cell lines. Optimization may be necessary. If so, vary DNA (µg): Lipofectamine2000 (µL) ratios from 1:0.5 to 1:5. If using a different transfection reagent, please consult the product manual.
3. Possible Cause: Not enough plasmid DNA used for dilution or complex formation
   Suggested Solution: Verify concentration using a second method or check the DNA for degradation. Determine DNA concentration by performing A260/A280 readings on a spectrophotometer or by using the Quant-iT DNA Assays Kits (Q33130, Q33120).
4. Possible Cause: Plasmid DNA or siRNA used in transfection has degraded or is of poor quality
   Suggested Solution: Ensure that the plasmid DNA or siRNA used for transfection is of high quality. For plasmid DNA purification kits, we recommend using our PureLink HiPure Nucleic Acid Purification Kits.
5. Possible Cause: Cell density was not optimal
   Suggested Solution: Lipofectamine 2000 works best in cultures that are >90% at the time of transfection.
6. Possible Cause: Complexes were added to cells in serum-free medium
   Suggested Solution: Try using growth medium containing serum when performing transfections. Transfection performance is typically better when the cells are more viable. If you require serum-free conditions, test medium for compatibility with Lipofectamine 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.
7. Possible Cause: Inhibitors were present in medium
   Suggested Solution: Do not use antibiotics, EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium used to prepare DNA:transfection reagent complexes.
8. Possible Cause: Problems with assay used to measure efficiency or expression
   Suggested Solution: Use a reporter gene to measure transfection efficiency. A reporter gene control allows you to confirm expression.
9. Possible Cause: Promoter-enhancer on vector is not recognized by the cell type
   Suggested Solution: Verify that the promoter-enhancer on your vector construct is compatible with the target cell type.
10. Possible Cause: Cells have changed over time, or splitting conditions have changed
    Suggested Solution: If transfection performance suddenly declines, it may be because of the cells. We recommend splitting and plating cells on a consistent schedule and in a manner where the cells are never too sparse or too dense. Excessive passaging also decreases transfection performance. If this is the case, start a new vial of cells from liquid nitrogen.
11. Possible Cause: Transfection reagent stored improperly
    Suggested Solution: We recommend storing transfection reagents at 4°C. Freezing of transfection reagents, or storing them at room temperature, may decrease activity.


Find additional tips, troubleshooting help, and resources within our Lipid-Based Transfection Support Center.

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Our phosphospecific ELISA kits have several advantages, including ease of use and increased sensitivity. Phosphospecific ELISA kits are typically 2-10 times more sensitive than western blots, so they are particularly useful for the detection of “low-expressing” proteins or for small sample sizes. In addition, with the use of the recombinant standards provided in the kit, phosphospecific ELISAs provide quantitative results without having to perform densitometry.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

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There are 2 main causes of poor ELISA standard curves. First, the recommended method for solubilizing the kit standard may not have been followed. The standard should be reconstituted according to the directions indicated on the label, using the standard diluent provided in the kit. No other diluent should be used. The vial should then be swirled or mixed gently and then allowed to sit for 10 minutes at room temperature to ensure complete solubilization. This concentrated standard solution should be used within 1 hour of reconstitution. Also, it should be mixed gently again before preparing the dilutions in standard diluent according to the instructions provided in the product manual. Leftover standard can usually be stored frozen in small aliquots, unless specified otherwise in the product manual.

The second common reason for poor standard curves is that the HRP conjugate was not diluted correctly. The 100X HRP conjugate solution contains 50% glycerol, which makes it very viscous and difficult to pipet accurately. Here is what we suggest to solve this problem: First, let the vial of HRP conjugate come to room temperature. Then, stir it gently with a clean pipet tip to make sure that it is homogeneous. Use only the separate HRP conjugate diluent provided in the kit to dilute it, and follow the dilution instructions provided in the manual.

The key to diluting the HRP conjugate is to make sure that it is pipetted correctly. You should test that your pipettor accurately aspirates and dispenses the volume of the conjugate-glycerol mixture that is required. If possible, this pipettor should be calibrated so it is accurate and reliable. When you aspirate the viscous conjugate solution, it may take 5-10 seconds for the desired amount to enter the pipet tip. Before transferring the conjugate to the appropriate HRP diluent, make sure that the outside of the pipet tip is dry by wiping it with a lab tissue (e.g., Kimwipes tissue), taking care to ensure that the contents inside the tip do not get absorbed by the tissue. Pipet the conjugate into the diluent, and then rinse out the tip by pipetting up and down several times. It is important to get every last bit of conjugate out of the tip. Next, seal the container and mix it gently but thoroughly by rocking it or turning it upside down. This is crucial because the glycerol carries the conjugate quickly down to the bottom of the tube. If the diluted conjugate is not mixed adequately, the concentration of the HRP conjugate will not be what is required.

Once the HRP conjugate is diluted and mixed gently but well, use it within 15 minutes. Remember that the HRP conjugate diluent is the only acceptable diluent for the HRP conjugate. The diluted HRP conjugate should not be saved because the HRP activity is labile, and it should never be stored and reused.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

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Both types of ELISA kits capture total protein, regardless of its phosphorylation state, within the wells of a plastic 96-well plate. This is done by coating the wells with a “pan-antibody” that does not distinguish between the phosphorylated and non-phosphorylated forms of a protein and does not block the phosphorylation site to be studied. In addition, a phosphospecific ELISA kit quantifies the amount of that same protein that is phosphorylated on one or more specific amino acids. Instead of a second pan-antibody for detection, this assay uses an antibody that specifically recognizes an epitope that is only present on a protein when it is phosphorylated specifically (i.e., it is phosphospecific).

We recommend running the total and phosphospecific ELISAs simultaneously with the same samples. If this is not possible, make sure to test the same samples with both kits as soon as possible.

Find additional tips, troubleshooting help, and resources within our Antibodies and Immunoassays Support Center.

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