Carbonate buffer

Manufactured by Merck Group

Sourced in United Kingdom, Spain

Carbonate buffer is a chemical solution used in laboratory settings to maintain a specific pH range. It is a mixture of sodium carbonate and sodium bicarbonate, which together create a buffering system that helps stabilize the pH of the solution. The primary function of carbonate buffer is to provide a controlled, consistent pH environment for various experimental and analytical procedures.

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Carbonate-bicarbonate buffer (61 citations) Carbonate-bicarbonate coating buffer (14 citations) Carbonate-bicarbonate buffer pH 9.6 (8 citations) Sodium carbonate buffer (5 citations) Carbonate buffer pH 9 (2 citations) 0.05-M carbonate/bicarbonate buffer (2 citations)

14 protocols using carbonate buffer

SARS-CoV-2 Spike Protein ELISpot Assay

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ELISpot assays were performed on isolated PBMCs before and after vaccination. MultiScreen-IP Filter plates (Millipore) were coated with 10 μg/mL of human anti-IFN-γ coating antibody (clone 1-D1K, Mabtech) in a carbonate buffer (Sigma-Aldrich) and stored at 4 °C overnight. The coated plates were washed three times with PBS and blocked with R10 media for a minimum of 1 h at 37 °C. After the blocking. 1.25 × 10⁵ PBMCs were added into each well as per the assigned layout. PBMCs were stimulated with a SARS-CoV-2 S1 pool (Wuhan strain) of 15-mer, with 11 amino acid overlap containing amino acid sequence 1–692 (PepTivator^®) at a final concentration of 1 µg/mL. Each assay was performed in duplicate and incubated for 16–18 h at 37 °C with 5% CO₂. Plates were developed by washing them six times with PBS/T, followed by the addition of 1 μg/mL of anti-IFN-γ detector antibody (7-B6-1-Biotin, Mabtech) to each well. After a 2-h incubation, plates were washed again, and 1:1,000 SA-ALP was added for 1 h at RT. After a final wash step, plates were developed using BCIP NBT-plus chromogenic substrate (Mabtech). ELISpot plates were counted using an Immunospot Microanalyzer (Cellular Technology Limited). Responses were averaged across duplicate wells, and the mean response of the unstimulated (negative control) wells were subtracted. Results are shown as SFCs/10⁶PBMCs.

Pinpathomrat N., Intapiboon P., Seepathomnarong P., Ongarj J., Sophonmanee R., Hengprakop J., Surasombatpattana S., Uppanisakorn S., Mahasirimongkol S., Sawaengdee W., Phumiamorn S., Sapsutthipas S., Kongkamol C., Ingviya T., Sangsupawanich P, & Chusri S. (2022). Immunogenicity and safety of an intradermal ChAdOx1 nCoV-19 boost in a healthy population. NPJ Vaccines, 7, 52.

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Biotin-labelled Pgp3 ELISA Protocol

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Biotin-labelled Pgp3 was produced using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific). Optimised assay conditions were determined by checkerboard titrations, as previously described [9 (link)]. Maxisorp microtitration plates (Nunc) were coated with unlabelled Pgp3 with bovine serum albumin (BSA) in carbonate buffer, pH 9.6 (Sigma) at 4°C. The protein-coated wells were blocked and stabilised by dilution buffer (PBS with 0.05% Tween-20 (PBST) (Sigma) with 1% Hammersten casein (GE Healthcare)) containing 5% sucrose (Sigma). Bound protein was incubated with either Pgp3 antibody-positive or negative defibrinated plasma [20 (link)] (25μl) diluted in dilution buffer (75μl) containing BSA at 37°C. After washing with PBST, biotinylated Pgp3 was added, incubated at 4°C, washed and incubated with horseradish peroxidase (HRP)-labelled streptavidin (Thermo Scientific). Finally, HRP activity was measured with TMB substrate (Biorad).
Assay cut-off was determined by receiver operating characteristic (ROC) analysis of absorbance (450–620nm) values on 505 paediatric samples and 342 samples from GUM patients, previously used to characterize our indirect ELISA [9 (link)]. As before, we determined specificity on the 494 samples from micro-immunofluorescence (MIF) assay-negative children [9 (link)].

Horner P.J., Wills G.S., Righarts A., Vieira S., Kounali D., Samuel D., Winston A., Muir D., Dickson N.P, & McClure M.O. (2016). Chlamydia trachomatis Pgp3 Antibody Persists and Correlates with Self-Reported Infection and Behavioural Risks in a Blinded Cohort Study. PLoS ONE, 11(3), e0151497.

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Competitive ELISA for Fentanyl Affinity

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Relative affinity was determined by competition ELISA. 96-well plates (Costar polystyrene high-binding plates, Corning) were coated overnight with fen-G4 conjugated to BSA, 50 ng/mL in a 50 mM carbonate buffer, pH 9.6 (Sigma). Plates were blocked with 1% gelatin for 1 h, washed with PBS-T, and free drug was loaded onto plates as competitor. Plates were incubated with 20 ng/mL HIS-tagged Fab for 2 h, washed, and incubated with Penta-His-biotin conjugate 1:5000 (Qiagen) overnight. Streptavidin-HRP 1:5000 (Thermo Fisher) was added, and plates were incubated for 1 h and washed. Fab bound to plates was measured using SigmaFAST OPD substrate (Sigma), and quantitated by absorbance at 492 nm on a microplate reader (Tecan). The fentanyl-hapten structure used for this particular assay differs from the structures presented in Figure S1. Specifically, we here used the “F3” hapten structure from Robinson et al., 2020,^{27 (link)} with the exact same BSA-conjugation methodology described in that publication.

Rajantie I., Ekman N., Iljin K., Arighi E., Gunji Y., Kaukonen J., Palotie A., Dewerchin M., Carmeliet P, & Alitalo K. (2001). Bmx Tyrosine Kinase Has a Redundant Function Downstream of Angiopoietin and Vascular Endothelial Growth Factor Receptors in Arterial Endothelium. Molecular and Cellular Biology, 21(14), 4647-4655.

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Quantifying Arginase Activity in Plasma

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Plasma from HDs and SFT patients was tested for arginase activity by measuring the production of L-ornithine from L-arginine, as previously reported (Rodriguez et al, 2004 (link)). In brief, a mix of 25 μl of plasma and 25 μl of buffer (Tris-HCl 50 nM pH 7.5 plus 10 mM MnCl₂, Sigma-Aldrich) was heated at 55 °C for 20 min. Then, 150 μl carbonate buffer (100 mM; Sigma-Aldrich) and 50 μl L-arginine (100 mM; Sigma-Aldrich) were added and the mix incubated at 37 °C for 20 min. The hydrolysis of L-arginine was stopped with 750 μl of glacial acetic acid. In all, 250 μl of ninhydrin solution (2.5 g ninhydrin (Sigma-Aldrich); 40 ml H₃PO₄ 6 M; 60 ml glacial acetic acid) was added followed by incubation at 95 °C for 1 h. The amount (nmol) of L-ornithine was determined measuring the absorbance at 570 nm.

Tazzari M., Negri T., Rini F., Vergani B., Huber V., Villa A., Dagrada P., Colombo C., Fiore M., Gronchi A., Stacchiotti S., Casali P.G., Pilotti S., Rivoltini L, & Castelli C. (2014). Adaptive immune contexture at the tumour site and downmodulation of circulating myeloid-derived suppressor cells in the response of solitary fibrous tumour patients to anti-angiogenic therapy. British Journal of Cancer, 111(7), 1350-1362.

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Quantifying Arginase Activity in Granulocytes

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Granulocyte cell extracts from patients and controls were tested for arginase activity by measuring the conversion of a known quantity of L-arginine to L-ornithine and urea. Briefly, 25µg of protein from cell lysates were added to 25 µl of Tris-HCl (50 mM; pH 7.5) containing 10 mM MnCl₂. This mixture was heated at 55–60°C for 10 min to activate arginase. Then, a solution containing 150 µL carbonate buffer (100 mM) (Sigma) and 50 µl L-Arg (100 mM) was added and incubated at 37°C for 20 min. The hydrolysis reaction from L-Arg to L-ornithine was identified by a colorimetric assay after the addition of ninhydrin solution and incubation at 95°C for 1 h. In addition, the hydrolysis reaction from L-Arg to urea was detected with diacetyl monoxime (Sigma) and incubation at 95°C for 10 min.

Dimitriades V., Rodriguez P.C., Zabaleta J, & Ochoa A.C. (2014). Arginase I levels are decreased in the plasma of pediatric patients with Atopic Dermatitis. Annals of allergy, asthma & immunology : official publication of the American College of Allergy, Asthma, & Immunology, 113(3), 271-275.

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COVID-19 Antibody Detection Assay

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For the COVID-19 assay in microtiter plates, 10 μg/mL
of SARS-CoV2 S1 antigen diluted in carbonate buffer (Sigma-Aldrich,
UK) was coated onto the plate (Nunc Immunosorp) in duplicates and
incubated overnight at 4 °C. The plate was washed three times
with PBS-Tween 20 and blocked three times with Superblock. Patient
samples were diluted as described above and incubated for 1 h at RT.
Following four washes with PBS-Tween 20, the secondary antibody was
diluted as described above and incubated for 1 h at RT. Finally, following
four washes with PBS-T, the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine
(TMB) (Europa Bioproducts, UK) was added and color development was
stopped by the addition of an equal volume of 0.25 M sulfuric acid.
Absorbance was read at 440 nm against a reference read at 700 nm.
For each set of duplicates, the average and the standard deviation
were calculated.

Reis N.M., Needs S.H., Jegouic S.M., Gill K.K., Sirivisoot S., Howard S., Kempe J., Bola S., Al-Hakeem K., Jones I.M., Prommool T., Luangaram P., Avirutnan P., Puttikhunt C, & Edwards A.D. (2021). Gravity-Driven Microfluidic Siphons: Fluidic Characterization and Application to Quantitative Immunoassays. ACS Sensors, 6(12), 4338-4348.

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Fluorescent Labeling of Nanoparticles for Macrophage Uptake

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The NPs used in the ex-vivo experiments were either 100 nm or 500 nm U-PS (Sigma-Aldrich) or A-PS (Sigma-Aldrich) and were labelled with green fluorescent dye (Fluorescein isothiocyanate (FITC), Sigma-Aldrich) dissolved in carbonate buffer (Sigma-Aldrich) at pH 9.6 at a conc of 1 mg/mL. The FITC was then diluted 1:10 in nanopure water containing the nanoparticles and incubated at room temperature for 1 hour with rotation. The NPs were centrifuged for 10 min at 20,937g and washed twice with nanopure water. We have previously shown that the coupling of FITC to the NPs by this methodology only provides minimal leaching at pH 7.4 and 4.0, to mimic the conditions of the extracellular and endosomal environments, respectively (Kendall et al., 2013 (link)). Alveolar macrophages were isolated from C57Bl/6 and SP-A^−/− mice as described above, the cells were washed and then incubated with NPs (1:5; 25,000 cells:125,000 NPs), after 5 min sonication, at 37°C for 30 min. The cells were centrifuged at 300g for 10 min and washed 3 times with RPMI Gibco) to remove excess beads and re-suspended in cytofix (BD bioscience) containing 0.2% trypan blue, to quench extracellular fluorescence as previously described (Hartshorn et al., 1994 (link)). The fixed cells were then analysed by fluorescence-activated cell sorting (FACS).

McKenzie Z., Kendall M., Mackay R.M., Whitwell H., Elgy C., Ding P., Mahajan S., Morgan C., Griffiths M., Clark H, & Madsen J. (2015). Surfactant protein A (SP-A) inhibits agglomeration and macrophage uptake of toxic amine modified nanoparticles. Nanotoxicology, 9(8), 952-962.

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Fluorescent Labeling of Recombinant Proteins

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Labelling of recombinant Hsp70 protein, bovine serum albumin (BSA), and the cmHsp70.1 mAb which recognizes mHsp70 (46 (link)) was performed according to our standardized protocol. Briefly, freshly prepared carbonate buffer (1 M) was added to the protein solution (1 mg/mL of phosphate-buffered saline (PBS)) at a ratio of 1:10 v/v followed by 50 μL of FITC (10 mg/mL in 0.1 M carbonate buffer, Sigma). The solution was incubated in the dark with gentle overnight shaking at 4°C. After dialysis using Slide-A-Lyzer™ Dialysis Cassettes (ThermoFisher Scientific), the protein concentration and dye/protein ratio were calculated using a microplate spectrophotometer (PerkinElmer). To avoid bacterial contamination, 0.02% w/v sodium azide was included in the solution, and the final stock was stored at 4°C, in the dark. Labelling of proteins using the Alexa Fluor™ 555 Labelling and Detection Kit (ThermoFisher Scientific) was conducted.

Bashiri Dezfouli A., Yazdi M., Benmebarek M.R., Schwab M., Michaelides S., Miccichè A., Geerts D., Stangl S., Klapproth S., Wagner E., Kobold S, & Multhoff G. (2022). CAR T Cells Targeting Membrane-Bound Hsp70 on Tumor Cells Mimic Hsp70-Primed NK Cells. Frontiers in Immunology, 13, 883694.

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LCMV Antibody ELISA in Mouse Sera

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Anti-LCMV antibodies in mouse sera were measured on Nunc PolySorp 96 well plates (Thermo Fisher) coated with sonicated cell lysate from LCMV-infected BHK-21 cells diluted in carbonate buffer (Sigma). Alkaline phosphatase-conjugated anti-mouse IgG antibodies (Southern Biotech) and phosphatase substrate (Sigma) were used for detection. ODs were read at 405nm on a SpectraMax Microplate Reader (Molecular Devices). Two-fold serial dilutions of the serum from a WT day 14-infefcted mouse was used to calculate relative ELISA units.

Song W., Antao O.Q., Condiff E., Sanchez G.M., Chernova I., Zembrzuski K., Steach H., Rubtsova K., Angeletti D., Lemenze A., Laidlaw B.J., Craft J, & Weinstein J.S. (2022). Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers. Immunity, 55(2), 290-307.e5.

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Fluorescent Nanoparticle Uptake by Alveolar Macrophages

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The NPs used in the ex vivo experiments were either 100 nm or 500 nm U-PS (Sigma-Aldrich) or A-PS (Sigma-Aldrich) and were labelled with green fluorescent dye (fluorescein isothiocyanate (FITC), Sigma-Aldrich) dissolved in carbonate buffer (Sigma-Aldrich) at pH 9.6 at a concentration of 1 mg/mL. The FITC was then diluted 1:10 in nanopure water containing the NPs and incubated at room temperature for one hour with rotation. The NPs were centrifuged for 10 min at 20 937 g and washed twice with nanopure water. We have previously shown that the coupling of FITC to the NPs by this methodology only provides minimal leaching at pH 7.4 and 4.0, to mimic the conditions of the extracellular and endosomal environments, respectively (Kendall et al., 2013 (link)). AMs were isolated from C57Bl/6 and SP-A^−/− mice as described above, the cells were washed and then incubated with NPs (1:5; 25 000 cells:125 000 NPs), after 5 min sonication, at 37 °C for 30 min. The cells were centrifuged at 300 g for 10 min and washed three times with RPMI (Gibco) to remove excess beads and re-suspended in cytofix (BD Biosciences) containing 0.2% trypan blue to quench extracellular fluorescence as previously described (Hartshorn et al., 1994 (link)). The fixed cells were then analysed by fluorescence-activated cell sorting (FACS).

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