Eight-week-old male specific pathogen-free (SPF) C57BL/6 mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). The mice were randomly divided into the following four groups (n=6 per group): normal control (NC) group (0.9% saline); the alcohol group [30% vol/vol alcohol 5 g/(kg·d) by gavage]; the butyrate group [butyrate (Sigma-Aldrich, USA) dissolved in 0.9% saline solution, gavage with 0.5 g/(kg·d)]; and the alcohol + butyrate group [30% vol/vol alcohol 5 g/(kg·d) by gavage, followed by butyrate 0.5 g/(kg·d) by gavage after a 1-hour interval]. All interventions were conducted continuously for 6 months. All of the mice were fed a normal chow diet, and were bred and maintained in our certified animal facility under a 12-hour light-dark cycle at 22 °C. The animal experiments were approved by the Ethics Review Committee of Southwest Medical University (No. 2020ZRQNA009). All experiments were performed in accordance with the approved guidelines and followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Butyrate
Manufactured by Merck Group
Sourced in United States, Germany, China, Netherlands
Butyrate is a laboratory product used to measure organic compound levels in various samples. It serves as a standard for analytical techniques, enabling the quantification and identification of other compounds.
Automatically generated - may contain errors
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121 protocols using butyrate
1
Long-term Alcohol and Butyrate Effects on C57BL/6 Mice
2
Butyrate Modulation of CIEC Proliferation
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CIECs were (ATCC Cell Bank, Shanghai, China) prepared with DMEM, including 10% fetal bovine serum (FBS) and 1% antibiotics. Next, different butyrate concentrations (0, 0.1, 0.5, 2 or 5 mM; Sigma-Aldrich, St. Louis, MO, USA) were added into the medium and incubated at 37 °C in 5% CO2 for 24 h. The proliferation level of the CIECs was determined by methyl thiazolyl tetrazolium (MTT) assay. We used the stimulation index (SI) to express the proliferative activity of CIECs. SI = OD570 (stimulated cells)/OD570 (unstimulated cells).
In addition, to explore the intracellular signaling pathway activated by butyrate, we added 10 μM LY294002 (an inhibitor of PI3k, MCE, Weehawken, NJ, USA), 2 μM GSK690693 (an inhibitor of AKT, MCE, Weehawken, NJ, USA), 5 μM CHIR99021 (an inhibitor of GSK-3β, MCE, Weehawken, NJ, USA), 20 μM 4-CMTB (an agonist of GPR43, MCE, Weehawken, NJ, USA), 5 μM AR420626 (an agonist of GPR41, GlpBio, Montclair, NJ, USA), 50 ng/mL PTX (an inhibitor of Gi, List Biological Laboratories, Campbell, CA, USA) and 10 μM Ym254890 (an inhibitor of Gq, Wako Pure Chemical Industries, Ltd. Osaka, Japan), respectively, into CIECs for 0.5 h before the addition of exogenous butyrate (0.5 mM, Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours later, CIECs were collected for western blot assay. Each assay used a repeat of six wells.
In addition, to explore the intracellular signaling pathway activated by butyrate, we added 10 μM LY294002 (an inhibitor of PI3k, MCE, Weehawken, NJ, USA), 2 μM GSK690693 (an inhibitor of AKT, MCE, Weehawken, NJ, USA), 5 μM CHIR99021 (an inhibitor of GSK-3β, MCE, Weehawken, NJ, USA), 20 μM 4-CMTB (an agonist of GPR43, MCE, Weehawken, NJ, USA), 5 μM AR420626 (an agonist of GPR41, GlpBio, Montclair, NJ, USA), 50 ng/mL PTX (an inhibitor of Gi, List Biological Laboratories, Campbell, CA, USA) and 10 μM Ym254890 (an inhibitor of Gq, Wako Pure Chemical Industries, Ltd. Osaka, Japan), respectively, into CIECs for 0.5 h before the addition of exogenous butyrate (0.5 mM, Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hours later, CIECs were collected for western blot assay. Each assay used a repeat of six wells.
3
Modulation of Colon Epithelial Cell Responses
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Mouse normal colon intestinal epithelial cells (BALB-5047, USA) were obtained from the RIKEN Cell Bank (Ibaraki, Japan). The cells were cultured in 96-well culture plates (5 × 106 cells/mL) and 12-well culture plates (5 × 105 cells/mL). Some LPS-treated cells (10 nM, Solarbio Ltd., Beijing, China) were treated with 2 μM TWS119 (a selective GSK-3β antagonist; MCE, Nanjing, USA; LPS + TWS-cells), 100 μM PDTC (an NF-κB antagonist; MCE, Nanjing, USA; LPS + PDTC-cells), or 5 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA; LPS + butyrate cells). After butyrate supplementation for 30 min, some LPS + butyrate cells were sequentially treated with 50 μM ITSA-1 (a nonselective HDAC3 agonist; MCE, New Jersey, USA; LPS + butyrate + ITSA-1-cells) or 5 nM Chetomin (a nonselective HIF-1α antagonist; Abcam, Cambridge, MA, USA; LPS + butyrate + Chetomin-cells). Each plate of treated cells was incubated for 24 h.
The cells from the 96-well culture plates were assessed for proliferation activity using an MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; Sigma, St. Louis, MO, USA) assay. The optical density was determined using a microplate reader (Model 680, Bio-Rad, St. Louis, MO, USA) equipped with a 570 nm wavelength filter. The cells from the 12-well culture plates were collected for LDH assessment and Western blotting. Each assay used a repeat of 8 wells.
The cells from the 96-well culture plates were assessed for proliferation activity using an MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; Sigma, St. Louis, MO, USA) assay. The optical density was determined using a microplate reader (Model 680, Bio-Rad, St. Louis, MO, USA) equipped with a 570 nm wavelength filter. The cells from the 12-well culture plates were collected for LDH assessment and Western blotting. Each assay used a repeat of 8 wells.
4
Activation of PPARγ in HT-29 Cells
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The human epithelial cell lines HT-29 were obtained from the American Type Culture Collection (ATCC, Rockville, MD), HT-29 cells were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 50 IU/mL penicillin, 50 μg/mL streptomycin and 10% heat-inactivated fetal calf serum (FCS) in a humidified 5% CO2 atmosphere at 37 °C. All culture media were supplied by Lonza. All agonists and inhibitors were dissolved in DMSO following the manufacturer’s recommendations. PPARγ activator TDZs: pioglitazone (Pio, 5 μM), rosiglitazone (Rosi, 10 μM), troglitazone (Tro, 5 μM) were from Cayman Chemicals and were used interchangeably due to their same level of activation on the used reporter system. MAPK kinase inhibitor: U0126 (MEK1/2) was purchased from Calbiochem. Butyrate was used at 2 mM except in the dose-response experiment where a range of concentrations from 0.5 to 8 mM was assessed for all the compounds tested (acetate, Butyrate, propionate, formate, lactate and succinate (Sigma).
5
Modulating Cecal Tonsil T Cell Proliferation
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T lymphocyte was obtained aseptically from cecal tonsil and cultured in RPMI 1640 medium. Then, T lymphocyte was stimulated with ConA (20 μg/ml, Sigma, St. Louis, MO, USA) + butyrate (0.5 mM, Sigma, St. Louis, MO, USA) and incubated at 37°C with 5% CO2 for 44 h. The proliferation level of T lymphocyte was determined by a methyl thiazolyl tetrazolium assay. We used the stimulation index (SI) to express the proliferative activity of T lymphocyte: SI = OD570 (stimulated cells)/OD570 (unstimulated cells).
In addition, cecal tonsil T lymphocytes were prepared with either 20 μM 4-CMTB (a GPR43 agonist, MCE, New Jersey, USA), 10 nM TSA (an HDAC3 antagonist, MCE, New Jersey, USA), 50 μM ITSA-1 (an HDAC3 agonist, MCE, New Jersey, USA), 10 μM Stattic (a STAT3 antagonist, MCE, New Jersey, USA), and 25 nM rapamycin (an mTOR antagonist, MCE, New Jersey, USA) for 30 min before the addition of ConA (20 μg/ml, Sigma, St. Louis, MO, USA) and butyrate (0.5mM, Sigma, St. Louis, MO, USA). The suspensions were incubated at 37°C with 5% CO2 for 44 h, and the optical density value was later determined. Each assay used a repeat of 6 wells.
In addition, cecal tonsil T lymphocytes were prepared with either 20 μM 4-CMTB (a GPR43 agonist, MCE, New Jersey, USA), 10 nM TSA (an HDAC3 antagonist, MCE, New Jersey, USA), 50 μM ITSA-1 (an HDAC3 agonist, MCE, New Jersey, USA), 10 μM Stattic (a STAT3 antagonist, MCE, New Jersey, USA), and 25 nM rapamycin (an mTOR antagonist, MCE, New Jersey, USA) for 30 min before the addition of ConA (20 μg/ml, Sigma, St. Louis, MO, USA) and butyrate (0.5mM, Sigma, St. Louis, MO, USA). The suspensions were incubated at 37°C with 5% CO2 for 44 h, and the optical density value was later determined. Each assay used a repeat of 6 wells.
6
Oral Administration of LGG and Butyrate
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LGG administration was performed as previously described.24 (link) Mice were orally inoculated with LGG (ATCC 53103) (Bayer) (5 × 109 CFU per 200 μl of saline) daily for 7 d and then orally once every 3 d until the end of the experiment according to the manufacturer’s instructions.
For butyrate administration, butyrate (Sigma-Aldrich) was dissolved in saline and diluted in drinking water at a final concentration of 6 mol/L for gavage. Drinking water containing same concentration of saline was prepared as control solution. butyrate group and control group were administered continuously by daily gavage of 150 μl of prepared butyrate solution or control solution for 8 d.
For butyrate administration, butyrate (Sigma-Aldrich) was dissolved in saline and diluted in drinking water at a final concentration of 6 mol/L for gavage. Drinking water containing same concentration of saline was prepared as control solution. butyrate group and control group were administered continuously by daily gavage of 150 μl of prepared butyrate solution or control solution for 8 d.
7
Alkaline Phosphatase Activity in Murine Stromal Cells
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ST2 murine bone marrow stromal cells (RIKEN Cell Bank, Tsukuba, Japan) were expanded in serum-containing DMEM (Sigma–Aldrich) and seeded at 3 × 105/cm2 for the experiments. ST2 cells were exposed to 20 μM of IL, BU, HMF, and ML or 10 μM butyrate (Sigma–Aldrich) for 7 days. butyrate served as a positive control for increasing alkaline phosphatase activity.31 (link) For histochemical staining of alkaline phosphatase, fixed cells were incubated with a substrate solution containing naphthol AS-TR phosphate (Sigma–Aldrich) and fast blue BB salt (Sigma-Aldrich).32 (link) Images were captured under a light microscope (Echorevolve microscope; Euromex, Arnheim, Netherlands).
8
Propionate and SCFA Effects on SFs
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Cultured SFs were treated for 24 h with 250 µM propionate or a physiological mixture of the SCFAs acetate, propionate, and butyrate (300 µM acetate, 100 µM propionate, 100 µM butyrate, all from Sigma-Aldrich, St. Louis, MO, USA). LPS stimulation of the SFs was achieved by adding 200 ng/mL LPS from E. coli to the growth medium for 24 h. In vitro treatment with SCFAs used concentrations that were previously shown to be nontoxic to cells [9 (link)].
9
Colonic Organoids: Butyrate Treatment and RNA-Seq
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Colonic organoids were cultured for 4 days and then treated with butyrate (Sigma-Aldrich, St. Louis, MO, USA) or without butyrate (control treatment) for 24 h in the following experiments. Total RNA was extracted from colonic organoids with RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was generated using SMART-Seq HT kit (Takara Bio, Kusatsu, Japan) and RNA-Seq libraries were constructed with Nextera DNA Flex Library Prep kit and Nextera DNA Unique Dual Indexes Set A (Illumina, San Diego, CA, USA) and sequenced on NovaSeq6000 (Illumina) at the University of Florida NextGen DNA Sequencing Core Facility. RNA seq analysis was performed using CLC genomics workbench (Qiagen).
10
Generation of Immature Dendritic Cells
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To generate DCs, CD14+ monocytes were positively selected from PBMCs of healthy donors and IgE‐mediated FA patients by immunomagnetic procedure (Miltenyi Biotec). Immature DCs were then obtained by culturing CD14+ cells at 106 cells/mL in RPMI 1640 (Invitrogen), 10% FCS (Lonza), 50 ng/mL GM‐CSF, and 1000 U/mL IL‐4 with or without butyrate (Sigma‐Aldrich) as previously described.31
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