DPBS, no calcium, no magnesium, 500 mL bottle - FAQs

你们推荐使用多大浓度的EDTA对培养在Gibco Essential 8培养基和玻连蛋白（VTN-N）中的细胞进行传代？

我们推荐您使用以不含钙或镁的Dulbecco氏磷酸缓冲盐溶液（DPBS）制备的 0.5 mM EDTA溶液。（货号14190-144（在欧洲，货号为14190-094））

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

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.

What is the shelf life of DPBS after it has been opened?

We cannot provide an official expiration date for open bottles as this depends on the customer's use. Once opened, Thermo Fisher Scientific can no longer guarantee the performance of any product. However, if the storage conditions are followed as recommended, the stated expiration date (36 months from the date of manufacture) should apply to opened bottles as DPBS is extremely stable.

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What is the shelf life of DPBS (Cat. No. 14190136)?

DPBS has a shelf life of 36 months from the date of manufacture, when stored as recommended (15-30 degrees C).

Does Diploid Growth Serum-Reduced Medium (SRM) contain L-glutamine?

The basal Diploid Growth Serum-Reduced Medium already contains 6 mM L-glutamine.

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Do I have to adjust my infection parameters when using Diploid Production Serum-Free Medium (SFM)?

We recommend evaluating performance with Diploid Production Serum-Free Medium using current conditions, however, multiplicity of infection, time of infection, and time of harvest may be different than with conventional media. Closely monitor cells for cytopathic effect and evaluate viral titers at multiple time points.

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Can Diploid Growth Serum-Reduced Medium (SRM) be supplemented with different types of serum?

Diploid Growth Serum-Reduced Medium can be supplemented with Fetal Bovine Serum, Newborn Calf Serum, Donor Bovine Serum with Iron, or Bovine Serum.

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Which cell lines can I culture in Diploid Growth Serum-Reduced Medium (SRM)?

Diploid Growth Serum-Reduced Medium has been evaluated with fibroblast cell lines, including MRC-5, WI-38, IMR-90, BS-2, and CEF cells.

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How long can I use Diploid Growth Serum-Reduced Medium (SRM) supplemented with Diploid Growth Supplement?

Once supplemented, the complete Diploid Growth Serum-Reduced Medium must be used within 4 weeks.

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Can I freeze or thaw my cells in Diploid Growth Serum-Reduced Medium (SRM)?

Most diploid cell lines can be frozen and thawed in Diploid Growth Serum-Reduced Medium. Consult the Diploid Growth Serum Reduced Medium User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019015_DiploidGrowthSerumReducedMedium_UG.pdf) for additional information.

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How do I adapt my cells to Diploid Growth Serum-Reduced Medium (SRM)?

Cells growing in medium with serum can be transferred directly to Diploid Growth Serum-Reduced Medium without adaptation. Continue to monitor and passage cells for 2-3 passages until consistent growth is achieved. Cultures may benefit from supplementation with 1-2% serum.

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Can I get a specific value for the conductivity for your DPBS products?

Sorry, we do not test the conductivity of our DPBS products and hence cannot provide a value or range.

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Are your DPBS products DNase/RNase-free? Can I use them for RNA work?

Our DPBS products are for cell culture use and have not been tested for DNase/RNase activity. We do not recommend using these products for RNA work.

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What is the specific gravity of DPBS (without calcium and magnesium)?

The specific gravity of this product is approximately 1.006 kg/L.

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What is the overall molarity of DPBS (without calcium and magnesium)?

The overall molarity of this solution is 19.66. This is calculated by taking the sum of the dry weights and dividing it by the sum of the formula weight.

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Is a precipitate in DPBS (without calcium and magnesium) normal?

No, this is not normal and if there is a precipitate in the product, it cannot be used. If the precipitate will not go back into solution, please contact Technical Support at techsupport@thermofisher.com for a replacement.

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What concentration of EDTA is recommended for passaging cells cultured in Gibco Essential 8 Medium and on VTN-N?

We recommend 0.5 mM EDTA prepared in Dulbecco's Phosphate-Buffered Saline (DPBS) without calcium or magnesium (Cat. No. 14190-144; (in Europe, Cat. No. 14190-094)).

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