Product FAQ

Can I install ChAS 3.1 on my AGCC workstation computer?

Answer

Due to the amount of memory that ChAS 3.1 needs to operate, Affymetrix™ VERY STRONGLY recommends that you DO NOT install the ChAS 3.1 on production AGCC computers being used for scanning and operating fluidics systems.

Answer Id: E13870

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Product FAQ

What versions of NetAffx™ analysis files are compatible with ChAS 3.1?

Answer

NA33 (Hg19) is only compatible in ChAS 3.1. ChAS 3.1 automatically prevents you from selecting an incompatible NetAffx™ analysis file version for analysis or when viewing analysis results.

Answer Id: E13871

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Product FAQ

Can I open two files that have been analyzed with different NetAffx™ versions at the same time in the ChAS 3.1 browser?

Answer

The ChAS 3.1 browser allows for loading of different NetAffx versions at the same time (only if the versions are from all the same reference and genome builds).

The ChAS 3.1 browser cannot load NetAffx versions from different genome builds (Hg18 and Hg19) or from an external reference version (dbSNP build 135 and dbSNP build 132).

Answer Id: E13872

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Product FAQ

Is it possible to determine cytokine concentration in samples with low protein concentrations, such as cerebral spinal fluid (CSF)?

Answer

To assay cytokines in samples with low protein concentrations with our EASIA™ (direct ELISA) kits, one thing to consider is the effect of “matrix”. Matrix is the liquid environment which surrounds the cytokine. The composition of the matrix can influence the results of the ELISA. The measurement of cytokines with low protein concentrations such as CSF, tissue culture supernatant, broncheolar alveolar lavage fluid, and urine, may produce results which are not truly comparable with a standard curve generated with serum as the matrix. These matrices with low protein concentration should be modified so that they more closely resemble the matrix used to generate the standard curve. We have found that addition of just a single protein component to samples with low protein matrices, such as adding bovine serum albumin, is not sufficient to cause the matrix to resemble that of the standard curve. We suggest several methods to adjust the matrix of samples so that it more closely resembles that of the standard curve. The first method is to dilute the sample 1:4 (one part sample plus three parts diluent) with the diluent provided in the kit (usually Standard Zero). Alternatively, a pool of serum from healthy volunteers may be used in place of the diluent. If pooled serum is used, it is important that the concentration of the cytokine to be measured first be quantitated in this serum and the appropriate correction should be made. An example is given below:

If the pooled serum is found to have 10 pg/mL of the cytokine of interest and cytokine level in the sample diluted 1 part sample in 3 parts pooled serum is measured to be 50 pg/mL, the calculated concentration in the sample is 50 - (10 x 3/4) = 42.5 pg/mL, then correcting for the 1:4 dilution, 42.5 pg/mL x 4 = 170 pg/mL.

A second method for increasing the matrix protein in samples is designated the Point-0-Cytokine (or Point-0-Receptor, depending on the kit) method. This method should be considered for use with samples that have low cytokine concentrations in a matrix with a low protein concentration. The presence of the cytokine at low concentrations does not permit the researcher to dilute the sample 1:4 because the cytokine concentration in the diluted sample would be below the level of detectability by the assay. The use of the Point-0-Cytokine vials allows the researcher to increase the concentration of the protein without diluting the samples. Each vial of Point-0-Cytokine contains lyophilized serum protein. To use the Point-0-Cytokine vials, the researcher simply adds 500 microliters of the low protein sample to the contents of the vials, and allows the serum proteins to rehydrate. Many of the EASIA™ kits require Point-0-Cytokine vials for low protein, low cytokine containing samples. Other kits require Point-0-Receptor vials which are used in the same manner as the Point-0-Cytokine. CSF requires addition of PT0 cytokine or PT0 receptor, or the normal procedure can be followed, depending on the kit.

Answer Id: E5142

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Product FAQ

What is Mendelian error check? Is it available for all arrays?

Answer

The Mendelian error check analysis provides two key points of information:

-Are the input samples related (e.g., mother-child, father-child)?
-Do any chromosomes have an elevated occurrence of Mendelian errors?
The Mendelian error checking feature is for CytoScan™ arrays only.

Answer Id: E13873

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Product FAQ

What is a ΔRn value? What does it mean?

Answer

ΔRn values represent the reporter dye fluorescent signal normalized to that of the Passive Reference dye (e.g. ROX dye when using Applied Biosystems™ reagents) for a given Real-Time PCR reaction. The ΔRn value is the Rn fluorescence value of the probe minus the Rn value for the reference dye. This reliably calculates the magnitude of the signal generated by the given set of PCR conditions.

Answer Id: E1388

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Product FAQ

What is angiogenesis?

Answer

Angiogenesis-the formation of new blood vessels from existing vasculature-is an integral part of both normal and pathological processes. It is required for tumor growth and metastatic spread, and as a result is a hot research area within oncology. This complicated process results from the input of multiple signaling pathways.

During angiogenesis, endothelial cells disrupt the surrounding basement membrane, migrate toward an angiogenic stimulus, proliferate to form the new vessel, and reorganize to create the necessary three dimensional vessel structure. In vitro assays are widely used to study these functions in the presence of either angiogenic or antiangiogenic agents.

Answer Id: E12021

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Product FAQ

Does Thermo Fisher Scientific offer an ELISA kit for the detection of IL-8 in rat and mouse?

Answer

After much effort, the search for an IL-8 homologue in rats and mice has been unsuccessful. This has led many researchers to conclude that the role of IL-8 in rats and mice is assumed by other chemokines (Broaddus, VA and Hebert, CA, 1996). However, it has been demonstrated that when an anti-human IL-8 antibody was given intratracheally to rats, the measure of lung inflammation and injury were significantly attenuated (Mulligan, MS et al, 1993). In addition, the authors obtained positive immunostaining with the anti-IL-8 antibody on rat pulmonary artery endothelial cells stimulated by TNF-alpha and in the rat lung after immune complex deposition. The authors were unable to determine what cytokine was being neutralized by the antibody. CINC-1 (a rat CXC chemokine and homologue of human MGSA/GRO) was ruled out since the antibody utilized was shown not to cross-react with that chemokine. These data suggest that a rat IL-8-like molecule which is distinct from CINC-1 exists and plays an important role in neutrophil recruitment.

In addition to CINC-1, three other rat CXC chemokines have been identified: CINC-2 alpha, CINC-2 beta, and CINC-3 (GRO beta/MIP-2) (Richmond, A and Shattuck, RL, 1996; Nakagawa, H et al., 1994). To date, the IL-8 homologue has not been identified, but recent data from Lee, J et al. suggest that it may be MIP-2 in rats. The homolog in mice may be KC. We offer an ELISA kit for the detection of rat MIP-2: catalog number KRC1021 (one plate version) and KRC1022 (two plates version). Mouse KC ELISA kit is KMC1061 (one plate version).

References:
1. Broaddus, V.A. and C.A. Hebert (1996) The Role of IL-8 in Inflammatory Diseases, in Chemoattractant Ligands and Their Receptors. Horuk, R., Ed., CRC Press, New York, pp.16-18.
2. Mulligan, M.S., M.L. Jones, M.A. Bolanowski, M.P. Baganoff, C.L. Deppeler, D.M.Meyers, U.S. Ryan, and P.A. Ward (1993) Inhibition of lung inflammatory reactions in rat by antihuman IL-8 antibody. J. Immunol. 150:5585-5595.
3. Richmond, A. and R.L. Shattuck (1996) Melanoma growth stimulatory activity: Physiology, biology, structure/function, and role in disease, in Chemoattractant Ligands and Their Receptors. Horuk, R., Ed., CRC Press, New York, pp. 89-90.
4. Nakagawa, H., N. Komorita, F. Shibata, A. Ikesue, K. Konishi, M. Fujioka, and H. Kato (1994) Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization. Biochem. J. 301:545-550.
5. Lee, J., G. Cacalano, T. Camerato, K. Toy, M.W. Moore, and W.I. Wood (1995) Chemokine binding and activities mediated by the mouse IL-8 receptor. J. Immunol.155:2158-2164.

Answer Id: E5144

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Product FAQ

When designing my primers for the CRISPR oligonucleotide, what level or purity is recommended? And do the oligonucleotides need to be phosphorylated?

Answer

5′-nonphosphorylated desalted oligonucleotides are fine. However, HPLC or higher purity will increase the cloning efficiency of your double-stranded oligonucleotide into the Invitrogen™ GeneArt™ CRISPR Nuclease vector.

Answer Id: E10140

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Product FAQ

Can I export my data in text format?

Answer

Yes. With ChAS 3.1, you can now export table data in word (docx), pdf, text, or transfer to clipboard. You can export graphic view data in pdf, docx, and png formats.

Answer Id: E13874

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Product FAQ

With the new algorithm, do I need to make new reference model files for analysis?

Answer

While the reference model algorithm is unchanged, there are differences in file format used for reference model files. The reference model files provided by Affymetrix™ have been updated to work with ChAS 3.1. Users generating their own reference model file will need to regenerate the model file in ChAS 3.1 to be able to use it.

Answer Id: E13890

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Product FAQ

When should I use the new Poisson Plus algorithm in AnalysisSuite™ software v3.0?

Answer

You should use the Poisson Plus algorithm for projects that contain QuantStudio™ 3D Chips with target quantities >2,000 copies/µL.

Answer Id: E10375

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Product FAQ

How should I handle fresh hepatocytes once I receive them?

Answer

Upon receipt, fresh hepatocytes in suspension should be centrifuged at 4 degrees C immediately, following the instructions provided with the cells. Plated hepatocytes should be quickly removed from the box and the shipping medium replaced with fresh culture medium. The preservation medium used during shipment is meant to be kept cold; if the cells achieve a temperature higher than 8 degrees C, the shipping medium begins to have a toxic effect on the hepatocytes. We recommend that you let your plated cells acclimate overnight before beginning your experiment.

Answer Id: E12022

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Product FAQ

What procedures are recommended for the preparation of beta amyloid-containing samples?

Answer

Following are procedures which researchers have followed for handling beta amyloid in tissue and cell samples:

Kienlen-Campard, P, S. Miolet, B. Tasiaux, and J.-N. Octave (2002) Intracellular amyloid beta 1-42, but not extracellular soluble amyloid beta peptides, induces neuronal apoptosis. J. Biol. Chem. 277(18):15666-15670. These authors performed formic acid extraction of whole cells. Neurons (approximately 107) were scraped in ice cold PBS. Cell pellets were solubilized in 300 microliters of 70% formic acid. Formic acid-solubilized cell pellets were cleared by centrifuging at 16,000 x g for 5 minutes at 4 degrees C, and the supernatants were centrifuged at 21,000 x g for 20 minutes at 4 degrees C. The supernatants were vacuum dried and the resulting pellet was resuspended in 1 mL of alkaline carbonate buffer (2% Na2CO3, 0.1 N NaOH) and centrifuged 16,000 x g for 3 minutes at 4 degrees C. Protein concentration was determined by the BCA assay. For immunoprecipitation, 800 microliters of the supernatant was neutralized with 800 microliters of 1 M Tris-HCl, pH 6.8, and diluted 1:3 in H2O containing 10% FCS, 0.5% Triton™ X-100, and 0.5% Nonident P-40 (final concentrations). Beta amyloid 1-40 and Beta amyloid 1-42 concentrations were determined by ELISA using 100 microliters of neutralized extract.

Fagan, A.M., M. Watson, M. Parsadanian, K.R. Bales, S.M. Paul, and D.M. Holtzman (2002) Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer"s disease. Neurobiol. Dis. 9(3):305-318. This paper discusses analyzing beta amyloid by ELISA. This paper has an interesting variation on the measurement of beta amyloid: they looked at soluble beta amyloid and also insoluble beta amyloid. Here are the key points of their protocol: For analysis of total beta amyloid levels, half of the hippocampus from each animal was Dounce homogenized in 5 M guandine (50 nM Tris-HCl, pH 8.0) and beta amyloid ELISA was performed as described previously (Johnson-Wood, et al. 1997). For analysis of soluble beta amyloid, the other half of the hippocampus was homogenized on ice in 400 microliters TBS (25 mM Tris-HCl, 150 mM NaCl, 3 mM EDTA, pH 7.4) containing protease inhibitors (20 micrograms/mL aprotinin, and 10 micrograms/mL leupeptin). Homogenates were spun at 125,000xg in polyallomer tubes in a Sorvall RP100 AT4-406 rotor for 1 hour at 4 degrees C and levels of beta amyloid total in the resultant supernatant (defines as soluble Abeta total) were obtained by beta amyloid ELISA. Percentage of soluble Abeta total was defined as the soluble (TBS-extractable) value divided by the total tissue (guanidine-extractable) value.

For Brain Tissue Homogenization, Prepare the Following Solutions: 5 M guanidine HCl 50 mM TrisHCl, pH 8.0. Reaction Buffer BSAT-DPBS (Dulbecco’s phosphate buffered saline with 5% BSA and 0.03% Tween-20, see formulation below) supplemented with 1x Protease Inhibitor Cocktail from Calbiochem (catalog code 539131; contains AEBSF, aprotinin, E64, EDTA, and leupeptin). BSAT-DPBS Formulation 0.2 g/L KCl 0.2 g/L KH2PO4 8.0 g/L NaCl 1.150 g/L Na2HPO4 5% BSA 0.03% Tween-20 to 1 L ultrapure water and adjust the pH to 7.4. Protocol: Determine the wet mass of the mouse hemibrain (100 mg) or a human brain sample in an Eppendorf tube (Fisher K749520-0000). Add 8 x mass of cold 5 M guanidineHCl / 50 mM Tris-HCl (Solution "A", above) to the tube by 50 - 100 µL aliquots and grind thoroughly with a hand-held motor (Fisher K749540-0000) after each addition. (Optional: transfer the homogenate from above to 1 mL Dounce homogenizer and homogenize thoroughly.) Mix the homogenate at room temperature for 3 - 4 hours. The sample is stable and can be freeze-thawed many times at this stage. Dilute the sample with cold Reaction Buffer (Solution "B"). Centrifuge (microfuge or Sorvall) at 16,000 x g for 20 minutes at 4°C. Save the supernatant for the assay.

Answer Id: E5147

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Product FAQ

When I receive my CRISPR oligonucleotide, what should I resuspend it in?

Answer

Either nuclease-free water or TE buffer is fine, but please anneal using the appropriate Oligonucleotide Annealing Buffer for your annealing reaction. Subsequently, please serially dilute your primers in a final concentration of 1X Oligonucleotide Annealing Buffer.

Answer Id: E10141

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