Alkaline buffer solution

Manufactured by Merck Group

Sourced in United States

Alkaline buffer solution is a laboratory reagent that maintains a basic, or alkaline, pH environment. It is commonly used in various analytical and experimental procedures to control the acidity or basicity of solutions. The core function of this product is to provide a stable, consistent pH to facilitate chemical reactions and measurements.

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16 protocols using alkaline buffer solution

Cell Proliferation and Osteogenic Differentiation Assays

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Cell proliferation, i.e., total DNA amount of hASCs and hBMSCs, was determined quantitatively by CyQUANT^® cell proliferation assay (Thermo Fisher Scientific) after 7, 9, and 11 days of culture. Before cell lysis, time point images were taken with a Nikon eclipse TS100 inverted phase contrast microscope (Nikon, Tokyo, Japan), with 4 X light objective. The analysis was performed on samples collected in 0.1% Triton buffer (Sigma-Aldrich) according to the manufacturer’s protocol, as described previously [38 (link),40 (link)]. The fluorescence signal representing nucleic acid bound CyQUANT^® GR dye (Thermo Fisher Scientific) was measured at 480/520 nm with a microplate reader (Victor 1420 Multilabel Counter, Wallac, Turku, Finland).
Alkaline phosphatase activity denoting the early osteogenic differentiation of hMSCs was analyzed from the same cell lysates as cell proliferation, as previously described [38 (link),40 (link)]. Briefly, the samples were incubated 15 min at 37 °C in working solution consisting of 1:1 10.8 µM phosphatase substrate (Sigma-Aldrich), and 1.5 M alkaline buffer solution (Sigma-Aldrich), after which the reaction was halted with 1.0 M sodium hydroxide (Sigma-Aldrich). The chromogenic reactions of the samples were detected by measuring the absorbance at 405 nm with a Victor 1420 microplate reader (Wallac).

Hyväri L., Vanhatupa S., Ojansivu M., Kelloniemi M., Pakarinen T.K., Hupa L, & Miettinen S. (2023). Heat Shock Protein 27 Is Involved in the Bioactive Glass Induced Osteogenic Response of Human Mesenchymal Stem Cells. Cells, 12(2), 224.

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Evaluating Osteogenic Commitment of hASCs

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Alkaline phosphatase activity, an indicator of early osteogenic commitment, was quantitatively evaluated after 7 and 14 days of culture as described previously[22 (link)]. Briefly, 20 μl of cell lysate (the same as used for the proliferation assay) was combined with 90 μl working solution (alkaline buffer solution and phosphatase substrate mixed in equal amounts; both from Sigma-Aldrich) in a 96-well plate and incubated for 15 min at +37°C. The reaction was stopped by adding 50 μl 1 M NaOH (Sigma-Aldrich) and the absorbance was measured at 405 nm with Victor 1420 Multilabel counter (Wallac). To assess the late osteogenesis of hASCs cultured in glass extracts, Alizarin red S staining of calcium phosphate (CaP) mineral was conducted after 19 days of culture. Cells cultured on BaG discs could not be characterized with Alizarin red S due to the heavy background staining of the discs. In brief, cells were fixed with 4% paraformaldehyde (PFA) and stained with 2% Alizarin red S (pH 4.1–4.3; Sigma-Aldrich) solution for 10 min at room temperature. After washes, the samples were imaged. Finally, the dye was extracted with 100 mM cetylpyridinium chloride (Sigma-Aldrich) and the result was quantified by measuring the absorbances at 544 nm (Victor 1420 Multilabel counter).

Ojansivu M., Mishra A., Vanhatupa S., Juntunen M., Larionova A., Massera J, & Miettinen S. (2018). The effect of S53P4-based borosilicate glasses and glass dissolution products on the osteogenic commitment of human adipose stem cells. PLoS ONE, 13(8), e0202740.

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Quantifying ALP Activity in hBMSCs

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ALP activity in the seeded hBMSCs was measured after 7 and 21 days. Cell/scaffold constructs were sonicated with Triton-X 100 0.1% on ice followed by incubation at −80°C. Following two freeze-thawing cycles at −80°C, samples were incubated with working solution containing Sigma 104^® phosphatase substrate (Sigma-Aldrich) and alkaline buffer solution (Sigma-Aldrich). Stop solution (1 M NaOH) was added, and the absorbance measured at 405 nm.

Munir A., Døskeland A., Avery S.J., Fuoco T., Mohamed-Ahmed S., Lygre H., Finne-Wistrand A., Sloan A.J., Waddington R.J., Mustafa K, & Suliman S. (2019). Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration. Journal of Tissue Engineering, 10, 2041731419852703.

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Osteogenic Differentiation Analysis of ASCs and BMSCs

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ASCs and BMSCs in osteogenic and control media were fixed with paraformaldehyde 4% at day 3, 7 and 14 for ALP staining, using SIGMAFASTTM BCIP/NBT tablets (Sigma-Aldrich). Images of the staining were taken using the inverted microscope. For the ALP assay at day 14, ASCs and BMSCs were lysed in 0.1% Triton-X100 buffer (Sigma-Aldrich), followed by two freezing-thawing cycles at − 80 °C, after which 20 μl of cell lysate was added in 96-well plate and mixed with 90 μl of working solution containing Sigma 104^® phosphatase substrate (Sigma-Aldrich) and alkaline buffer solution (Sigma-Aldrich). After incubation at 37 °C for 15 min, 50 μl of NaOH (sodium hydroxide) was added to stop the reaction. Absorbance was measured at 405 nm using the microplate reader. ALP activity assay was presented relative to BMSCs cultured in control medium as control samples.

Mohamed-Ahmed S., Fristad I., Lie S.A., Suliman S., Mustafa K., Vindenes H, & Idris S.B. (2018). Adipose-derived and bone marrow mesenchymal stem cells: a donor-matched comparison. Stem Cell Research & Therapy, 9, 168.

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Alkaline Phosphatase Activity Assay

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The ALP activity was measured to study early differentiation of cells. Triton X-100 (0.1%) was added to the samples, followed by centrifugation at 13,000 rpm for 5 min at 4°C. The substrate solution (Sigma-Aldrich Co) and alkaline buffer solution (Sigma-Aldrich Co) were added to each sample at 37°C for 30 min. After the reaction stopped, 1 N NaOH solution was added to each sample. The samples were then transferred into 96-well plates, and the absorbance at 405 nm was measured using a microplate reader (Multiskan EX; Thermo Fisher Scientific).

Kang Y.G., Wei J., Shin J.W., Wu Y.R., Su J., Park Y.S, & Shin J.W. (2018). Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds. International Journal of Nanomedicine, 13, 1107-1117.

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Alkaline Phosphatase Activity Assay Protocol

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Lysates for alkaline phosphatase activity were obtained by adding 0.25 mL cold lysis buffer (1 mM MgCl2, 0.5% Triton-X100 in Alkaline Buffer Solution (Sigma)) per sample. After incubating for 1 h on ice, another 0.25 mL of cold lysis buffer was added. For the reaction, 100 µL lysate and 400 µL phosphatase substrate solution (20 mg p-nitrophenol phosphate (Sigma-Aldrich) in 1.25 mL Alkaline Buffer Solution and 3.75 mL ddH2O) were incubated in a water bath at 37 °C for 10 min. The reaction was stopped by adding a 500 µL stop solution (20 g NaOH, 37.22 g Na2EDTA in 500 mL ddH2O). Absorbance was read at 405 nm using the ELISA Zenith 200 (Anthos Labtec). ALP activity was calculated using serial dilutions of 4-nitrophenol solution (10 mM).

Novak R., Ahmad Y.A., Timaner M., Bitman-Lotan E., Oknin-Vaisman A., Horwitz R., Hartmann O., Reissland M., Buck V., Rosenfeldt M., Nikomarov D., Diefenbacher M.E., Shaked Y, & Orian A. (2022). RNF4~RGMb~BMP6 axis required for osteogenic differentiation and cancer cell survival. Cell Death & Disease, 13(9), 820.

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Quantification and Visualization of ALP Activity

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To quantify ALP activity, cell lysate previously produced was used. For that, 80 μl of cell lysate were combined with 20 μl of 1.5 M of Alkaline buffer solution (Sigma-Aldrich) and 100 μl of 4 mg/ml of phosphatase substrate (Sigma-Aldrich, USA). Then samples were incubated in the dark for 1 h at 37 °C. At this point, the reaction was stopped by adding 100 μl of 0.3 M of NaOH (PanReac AppliChem), and the absorbance was read at 405 nm using a microplate reader. The ALP activity was determined using a standard curve obtained from different dilutions of 4-Nitrophenol (Sigma-Aldrich) solution 10 mM, ranging from 0 to 250 μM.
ALP activity was also assayed by cytochemistry. For that, cell layers were fixed with 10% Neutral Buffered Formalin (ThermoFisher Scientific) for 20 min and permeabilized for 5 min with 0.1% v/v Triton X-100 (Sigma-Aldrich) in PBS. Afterward, cells were incubated for 30 min in Naphthol AS-MX phosphate/Fast Violet B salt (Sigma) at 37 °C and in the dark. Finally, hASCs were washed in PBS and observed using a microscope (Leica DM750).

Hurle K., Maia F.R., Ribeiro V.P., Pina S., Oliveira J.M., Goetz-Neunhoeffer F, & Reis R.L. (2021). Osteogenic lithium-doped brushite cements for bone regeneration. Bioactive Materials, 16, 403-417.

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Quantitative Alkaline Phosphatase Assay

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At day 10 after passage, ALP histochemistry was performed using diagnostic kit 85 (Sigma). ALP activity was determined using a colorimetric end point assay measuring the enzyme conversion of p-nitrophenyl phosphate (pNPP) to the yellowish product, p-nitrophenol (pNP), in the presence of ALP.1 (link) In brief, cells were rinsed with cold PBS, scraped into 1.5 ml Eppendorf tubes in 1ml of lysis buffer (10 mM Tris-HCl, 1 mM magnesium chloride, and 0.05% Triton X-100, pH7.5), and then homogenized on ice for 10 minutes. Afterwards, the resulting mixture was centrifuged at 12,000 rpm for 10 minutes at 4℃. The cell lysates (0.1 mL of the above supernatants) were mixed with 0.5 mL of pNP phosphate solution (Sigma) and 0.5 mL of alkaline buffer solution (Sigma). After incubation at 37℃ for 15 minutes, the above mixture was added to 1 mL of 0.5 N NaOH to stop the reaction and the absorbance was then measured spectrophotometrically at 405 nm. A standard curve of known concentration of pNP was generated concurrently and used to determine sample concentrations.

Lee H.M., Joo B.S., Lee C.H., Kim H.Y., Ock J.H, & Lee Y.S. (2015). Effect of Glucagon-like Peptide-1 on the Differentiation of Adipose-derived Stem Cells into Osteoblasts and Adipocytes. Journal of Menopausal Medicine, 21(2), 93-103.

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Quantitative Assessment of Osteoblastic Differentiation

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Alkaline Phosphatase (ALP) activity is a biochemical marker for osteoblast activity. Briefly, ALP production by the DPS-7 cells was assessed on days 7 and 14 of cell treatment with probiotic CM. First, 10⁵ hDPSC were cultured per well of a 24-well plate. After 7 and 14 days, cells were washed 3 times using Phosphate-Buffered Saline (PBS) solution. Cells were homogenized in 0.5 ml of RIPA buffer (pH=7.5, 0.05% Triton X-100, 1 mM MgCl₂, 10 mM Tris-HCl) using sonication and then centrifuged at 12,000 rpm for 10 min at 4°C. Cell degradation contents (0.1 ml of the floating contents) were mixed with 0.5 ml of p-Nitrophenol Phosphate (PNP) (Sigma-Aldrich, USA) solution and 0.5 ml of alkaline buffer solution (Sigma-Aldrich, USA). After incubation at 37°C for 15 min, 1 ml of 0.02 N NaOH was added to the mixture to stop the interaction and adsorption was measured at 405 nm using a spectrophotometer (Bio-Rad, USA) ^{8 (link)}. A standard curve of known PNP concentrations was plotted and used to calculate the concentration of the samples. The ALP activity was calculated as the ratio of the PNP nanomoles converted per min to milligrams of the total protein. Total protein was measured using the Bradford assay.

Amini F., Rezvani M.B., Bakhtiari R, & Tabatabaei Ghomsheh E. (2023). Effects of Dental Pulp Stem Cell Preconditioning on Osteogenesis using Conditioned Media of Probiotics Bacteria. Avicenna Journal of Medical Biotechnology, 15(2), 76-83.

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Quantifying Osteogenic Differentiation via ALP

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The activity of ALP was assessed as an early marker of osteogenic differentiation at day 6 after differentiation induction. Cells plated into 96-well plates were washed twice with PBS (phosphate buffered saline) and then fixed with 4% formaldehyde (Duksan, Gyeonggi-do, Korea) for 30 min at room temperature. ALP activity was determined colorimetrically by incubating cells with the substrate p-nitrophenyl phosphate (Sigma-Aldrich) diluted in a 1:15 ratio in alkaline buffer solution 1.5 M, pH 10.3 (Sigma-Aldrich) at 37 °C for 30 min. The absorbance was measured at 405 nm and normalized against each experimental group's MTT value with a Multiskan^TM GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Values were expressed as the fold change relative to control (undifferentiated) cells.

Dubon M., Lee S., Park J.H., Lee J.Y, & Kang D. (2021). The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC). International Journal of Medical Sciences, 18(7), 1580-1591.

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