Citrate buffer

Manufactured by Teknova

Sourced in United States

Citrate buffer is a commonly used buffer solution that maintains a specific pH range. It is formulated by combining citric acid and sodium citrate. The primary function of the citrate buffer is to maintain a stable pH environment for various biochemical and analytical applications.

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Lab products found in correlation

Alcohol solutions, by Merck Group (3 mentions) Dab solution, by Vector Laboratories (3 mentions) Goat anti rabbit igg conjugated to horseradish peroxidase, by Abcam (2 mentions) Rabbit anti human tenascin c monoclonal antibody, by Abcam (2 mentions) Cholesterol chol, by Merck Group (1 mentions) 1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (1 mentions) Squalene oil, by Merck Group (1 mentions) Polysorbate 80 tween 80, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Merck Group (1 mentions) Miniseq, by Illumina (1 mentions) C57bl 6j mice, by Jackson ImmunoResearch (1 mentions) Nanoassemblr spark, by Precision Nanosystems (1 mentions)

Close spelling variants of this product

RNase free 50 mM citrate buffer pH 3.0 (2 citations) QB Citrate Buffer (2 citations) 100 mmol l−1 citrate buffer (1 citations)

16 protocols using citrate buffer

Immunohistochemical Analysis of Tissue Microarrays

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The paraffin-embedded tissue arrays (BRN801a and BC081120, US Biomax, Rockville, MD) were dewaxed in xylene for 10 min twice and rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich, St. Louis, MO) (100% ethanol twice, 90% ethanol, 70% ethanol, 5 min each) to water. Antigen retrieval was done by boiling the slides for 15 min in a citrate buffer (Teknova, U.S) at pH 6.0. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 min after returning to room temperature. The TMAs were subsequently blocked with 2% BSA and incubated with monoclonal antibody (1:75) overnight at 4°C followed by incubation with a secondary antibody conjugated to horseradish peroxidase. Immunodetection was performed using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin counterstain was used to visualize nuclei. The protein expression level in each tissue section was assessed in non-necrotic areas of three separate microscopic fields of view under a magnification of 20× and was represented by the mean of the percentage of positive cells.

Nie S., McDermott S.P., Deol Y., Tan Z., Wicha M.S, & Lubman D.M. (2015). A quantitative proteomics analysis of MCF7 breast cancer stem and progenitor cell populations. Proteomics, 15(22), 3772-3783.

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Immunohistochemical Analysis of Tenascin-C Expression

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Immunohistochemical staining was performed using tissue microarray samples. The paraffin-embedded tissue arrays with 1.5 mm core diameter and 5 µm thickness were dewaxed in xylene for 10 min twice and rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich, St. Louis, MO) (100% ethanol twice, 90% ethanol, and 70% ethanol, 5 min each) to water. Then, the slides were boiled for 15 min in citrate buffer (Teknova, Hollister, CA) at pH 6.0 for antigen retrieval. After returning to room temperature, endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 min. The TMAs were then rinsed with water and PBS and subsequently blocked with 2% BSA and incubated with rabbit anti-human Tenascin-C monoclonal antibody (1:100 dilution, Abcam, Cambridge, MA) overnight at 4 °C followed by incubation with a goat anti-rabbit IgG conjugated to horseradish peroxidase (1:250 Abcam, Cambridge, MA). Immunodetection was performed using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin counterstain was used to visualize nuclei. The TNC expression level in each tissue section was assessed in non-necrotic areas of three separate microscopic fields of view under a magnification of 200× and was represented by the mean of the percentage of TNC⁺ cells. The results were confirmed by a pathologist.

Nie S., Gurrea M., Zhu J., Thakolwiboon S., Heth J.A., Muraszko K.M., Fan X, & Lubman D.M. (2014). Tenascin-C: A Novel Candidate Marker for Cancer Stem Cells in Glioblastoma Identified by Tissue Microarrays. Journal of Proteome Research, 14(2), 814-822.

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Immunohistochemical Analysis of Tenascin-C Expression

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Immunohistochemical
staining was performed using tissue microarray
samples. The paraffin-embedded tissue arrays with 1.5 mm core diameter
and 5 μm thickness were dewaxed in xylene for 10 min twice and
rehydrated through a series of alcohol solutions (200 proof, Sigma-Aldrich,
St. Louis, MO) (100% ethanol twice, 90% ethanol, and 70% ethanol,
5 min each) to water. Then, the slides were boiled for 15 min in citrate
buffer (Teknova, Hollister, CA) at pH 6.0 for antigen retrieval. After
returning to room temperature, endogenous peroxidase activity was
blocked with 3% H₂O₂ in methanol for 10 min.
The TMAs were then rinsed with water and PBS and subsequently blocked
with 2% BSA and incubated with rabbit anti-human Tenascin-C monoclonal
antibody (1:100 dilution, Abcam, Cambridge, MA) overnight at 4 °C
followed by incubation with a goat anti-rabbit IgG conjugated to horseradish
peroxidase (1:250 Abcam, Cambridge, MA). Immunodetection was performed
using DAB solution (Vector Laboratories, Burlingame, CA). Hematoxylin
counterstain was used to visualize nuclei. The TNC expression level
in each tissue section was assessed in non-necrotic areas of three
separate microscopic fields of view under a magnification of 200×
and was represented by the mean of the percentage of TNC⁺ cells. The results were confirmed by a pathologist.

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Lipid Nanoparticle Formulation for mRNA Delivery

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The ionizable cationic lipid O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraem-19-yl)-4-(N,N-dimethylamino)butanoate (DLin-MC3-DMA) was synthesized at AstraZeneca. The 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was obtained from Avanti Polar Lipids, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG₂₀₀₀) was obtained from NOF Corporation, and Cholesterol (Chol) was obtained from Sigma–Aldrich. Deuterated DSPC (d83) and Chol (d7) were obtained from Avanti Polar Lipids. The ³H-labeled DSPC was synthesized at AstraZeneca. Erythropoietin (EPO) mRNA (858 nucleotides) ARCA capped modified with 5-methylcytidine and pseudouridine was purchased from TriLink Biotechnologies. Polyadenylic acid (PolyA) was purchased from Sigma–Aldrich and had a molecular weight range between 600 and 4,000 bases according to analysis by agarose gel electrophoresis in formamide performed by the manufacturer. Hydrogeneous PBS (1 mM KH₂PO₄, 155 mM NaCl, and 3 mM Na₂HPO₄ 0.7H₂O, pH 7.4) was obtained from Life Technologies, whereas deuterated PBS was prepared with D₂O from Sigma–Aldrich. Citrate buffer was purchased from Teknova, and HyClone RNase free water was obtained from GE Healthcare Cell Culture.

Yanez Arteta M., Kjellman T., Bartesaghi S., Wallin S., Wu X., Kvist A.J., Dabkowska A., Székely N., Radulescu A., Bergenholtz J, & Lindfors L. (2018). Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles. Proceedings of the National Academy of Sciences of the United States of America, 115(15), E3351-E3360.

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Squalene Oil-based Emulsion Preparation

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Squalene oil, sorbitane trioleate (Span 85) and phosphate buffered saline (PBS) were obtained from Sigma Aldrich and polysorbate 80 (Tween 80) from Acros Organic. Millipore MilliQ deionized water was used and citrate buffer was acquired from Teknova. Chromatography column was obtained from Waters: Acquity UPLC (R) BEH C18 1.7 µm 2.1 × 50 mm.

Shah R.R., Taccone M., Monaci E., Brito L.A., Bonci A., O’Hagan D.T., Amiji M.M, & Seubert A. (2019). The droplet size of emulsion adjuvants has significant impact on their potency, due to differences in immune cell-recruitment and -activation. Scientific Reports, 9, 11520.

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Microfluidic Nanoparticle Formulation Protocol

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Nanoparticles were formulated with a microfluidic device as previously described^{20 (link)}. Nucleic acids (DNA barcodes and mRNA) were diluted in 10mM citrate buffer (Teknova). Lipid-amine compounds (7C1, cKK-E12, and cKK-E15), PEG-lipids (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]), cholesterols (cholesterol and 20α-hydroxycholesterol), and helper lipids (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-di-O-octadecenyl-3-trimethylammonium propane) were diluted in 100% ethanol. For mRNA screens, aVHH mRNA and DNA barcodes were mixed at a 10:1 mass ratio. All PEGs, cholesterols, and helper lipids were purchased from Avanti Lipids. Citrate and ethanol phases were combined in a microfluidic device by syringes (Hamilton Company) at a flow rate of 3:1.

Hatit M.Z., Lokugamage M.P., Dobrowolski C.N., Paunovska K., Ni H., Zhao K., Vanover D., Beyersdorf J., Peck H.E., Loughrey D., Sato M., Cristian A., Santangelo P.J, & Dahlman J.E. (2022). Species-dependent in vivo mRNA delivery and cellular responses to nanoparticles. Nature nanotechnology, 17(3), 310-318.

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Microfluidic Nanoparticle Formulation for Delivery

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Nanoparticles were formulated using a microfluidic device as previously described (26 (link)). Briefly, nucleic acids (mRNA, DNA barcodes, siRNA, and sgRNA) were diluted in 10 mM citrate buffer (Teknova) while lipid-amine compounds, alkyl-tailed PEG, cholesterol, and helper lipids were diluted in ethanol. For nanoparticle screens, Cre mRNA and DNA barcodes were mixed at a 10:1 mass ratio. Citrate and ethanol phases were combined in a microfluidic device by syringes (Hamilton Company) at a flow rate of 600 µL/min and 200 µL/min, respectively. All PEGs, cholesterol, and helper lipids were purchased from Avanti Lipids.

Sago C.D., Lokugamage M.P., Paunovska K., Vanover D.A., Monaco C.M., Shah N.N., Gamboa Castro M., Anderson S.E., Rudoltz T.G., Lando G.N., Munnilal Tiwari P., Kirschman J.L., Willett N., Jang Y.C., Santangelo P.J., Bryksin A.V, & Dahlman J.E. (2018). High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proceedings of the National Academy of Sciences of the United States of America, 115(42), E9944-E9952.

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Microfluidic Formulation of Barcoded LNPs

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Nanoparticles were formulated in a microfluidic device by mixing Cre mRNA, DNA, and LNP components^{[17 (link)]}. The nucleic acid was diluted in citrate buffer (Teknova). Nanoparticle materials were diluted in ethanol. The phases were mixed together via microfluidics. Each LNP was formulated to carry a distinct barcode; LNP1 carried Cre mRNA and DNA barcode 1, whereas LNP2 carried Cre mRNA and DNA barcode 2. LNP hydrodynamic diameter was measured using dynamic light scattering. All animal experiments were performed in accordance with the Georgia Tech IACUC. C57BL/6J mice were purchased from Jackson Laboratory. Mice were aged 5–8 weeks, and N = 3–4 mice per group were injected intravenously. Sequencing was performed on MiniSeq™ using Illumina protocols.

Paunovska K., Da Silva Sanchez A., Sago C.D., Gan Z., Lokugamage M.P., Islam F., Kalathoor S., Krupczak B.R, & Dahlman J.E. (2019). Nanoparticles containing oxidized cholesterol deliver mRNA to the liver microenvironment at clinically relevant doses. Advanced materials (Deerfield Beach, Fla.), 31(14), e1807748.

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Microfluidic Nanoparticle Formulation

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Nanoparticles were formulated in a microfluidic device by mixing DNA with lipomer, PEG, cholesterol, and a helper lipid, as previously described^{5 , 18 (link), 19 (link), 22 –27 (link), 42}. Nanoparticles were made with variable mole ratios of these constituents. The genetic drug (in this case, DNA barcode) was diluted in 10 mM citrate buffer (Teknova), and loaded into a syringe (Hamilton Company). The materials making up the nanoparticle (lipomer, cholesterol, PEG, and helper lipid) were diluted in 100% ethanol, and loaded into a second syringe. The citrate phase and ethanol phase were mixed together in a microfluidic device, at rates of 600 uL/min and 200 uL/min, respectively, to form LNPs. We used the following helper lipids: DOPE (Avanti Lipids, 850725), and DOPC (Avanti Lipids, 850375).

Paunovska K., Sago C.D., Monaco C.M., Hudson W.H., Castro M.G., Rudoltz T.G., Kalathoor S., Vanover D.A., Santangelo P.J., Ahmed R., Bryksin A.V, & Dahlman J.E. (2018). A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation. Nano letters, 18(3), 2148-2157.

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Microfluidic Synthesis of Lipid Nanoparticles

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Nanoparticles were formulated in a microfluidic device as previously described (26 (link)) by mixing a nucleic acid, an ionizable lipid, polyethylene glycol (PEG), cholesterol, and a phospholipid. Nanoparticles were made with variable mole ratios of these constituents. The mass ratios for all the constituents were as follows: RNA was 7.5:1 for LNP1 and 10:1 for LNP2. LNP1 consisted of the ionizable lipid cKK-E12, cholesterol (Avanti Lipids, 700000P), C₁₄PEG2K (Avanti Lipids, 880150P), and DOTAP (Avanti Lipids, 890890P) or NBD-DOTAP (Avanti Lipids, 810890P). LNP2 consisted of the ionizable lipid 7C1, cholesterol, C₁₄PEG2K, and DOPE (Avanti Lipids, 850725P). LNP3 consisted of the ionizable lipid cKK-E12, cholesterol, C₁₈PEG2K (Avanti Lipids, 880120P), and DOPE (Avanti Lipids, 850725P) or AF647-DOPE (Millipore-Sigma, 42247). Chemically modified mRNA (GFP or Cre) was purchased from TriLink. The mRNA was diluted in 10 mM citrate buffer (Teknova) and loaded into a syringe (Hamilton Company). The materials making up the nanoparticle (cKK-E12 or 7C1, cholesterol, PEG, and DOPE or DOTAP) were diluted in 100% ethanol and loaded into a second syringe. The citrate phase and ethanol phase were mixed together in a microfluidic device using syringe pumps.

Paunovska K., Da Silva Sanchez A., Foster M.T., Loughrey D., Blanchard E.L., Islam F.Z., Gan Z., Mantalaris A., Santangelo P.J, & Dahlman J.E. (2020). Increased PIP3 activity blocks nanoparticle mRNA delivery. Science Advances, 6(30), eaba5672.

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