This was undertaken essentially as previously described (Jiang et al., 2012 (link)). Briefly, loosely adherent BMDCs on a fluordish (Cat #FD35-100, Coherent Life Science), underwent synchronized phagocytosis (Jiang et al., 2012 (link)) with opsonised zFITCs on the heated stage of microscope stage of a Zeiss Axiovert 200M fluorescence microscope and the particle fluorescence was recorded over 60 min, at a rate of one image per minute (excitation 490 nm, emission 525 nm). In some instances, IAA94 (100 μM) (Cat #I117, Sigma-Aldrich), a cell permeable CLIC1 ion channel blocker, Chloroquine (Cat #C6628, Sigma-Aldrich) or DMSO (Cat #D2650, Sigma-Aldrich) were added to the fluordish. To convert the excitation ratio to pH, time lapse recordings over 45 min were carried out on BMDCs that had phagocytosed opsonised zFITC, incubated in a series of buffers from pH 4 to pH 8 which also contained bafilomycin A1 (100 nM), nigericin (10 µM), valinomycin (10 µM) and carbonyl cyanide m-chlorophenylhydrazone (10 µM) to disrupt membrane channel activity and allow equilibration of intracellular pH with that of the extracellular buffer. There was minimal if any photobleaching (Fig. S1A ). A polynomial equation from this data was then derived and used to convert the real time FITC intensity into pH units (Fig. S1B ).
Iaa94
Manufactured by Merck Group
Sourced in United States, Italy
IAA94 is a laboratory equipment product from Merck Group. It is a device designed for performing specific laboratory tasks. The core function of IAA94 is to assist in the analysis and processing of samples within a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Nigericin, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Anti nestin, by Abcam (2 mentions)
Anti gfap, by Abcam (2 mentions)
Metformin, by Merck Group (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Chloroquine, by Merck Group (1 mentions)
Poly da dt, by Merck Group (1 mentions)
Duolink in situ pla kit, by Merck Group (1 mentions)
Ac yvad cmk, by Bachem (1 mentions)
Flagellin fla st ultrapure, by InvivoGen (1 mentions)
Alexa fluor 488tm phalloidin, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Caspase 1, by Adipogen (1 mentions)
Phorbol 12, by Merck Group (1 mentions)
7 protocols using iaa94
1
Phagocytic Fluorescence Assay for Intracellular pH
2
Inflammasome Activation Assay Protocol
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LPS, ATP, nigericin, poly dA:dT, IAA94, and Duolink In Situ PLA kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ac-YVAD-cmk was obtained from Bachem (Torrance, CA, USA). The dye, 2′,7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM), MitoSOX, MQAE (N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide) and DAPI were obtained from Invitrogen (Waltham, MA, USA). Flagellin (FLA-ST Ultrapure) was obtained from InvivoGen (San Diego, CA, USA). Alexa fluor 488TM Phalloidin was purchased from ThermoFisher Scientific. Abs were purchased for the detection of NLRP3 (Adipogen, San Diego, CA, USA), ASC (Adipogen), IL-1β (R&D systems, Minneapolis, MN, USA), caspase-1 (Adipogen), β-actin (Santa-Cruz Biotechnology, Dallas, TX, USA), and CLIC1 (Proteintech, Rosemont, IL, USA) Na+-K+ ATPase (Cell Signaling Technology, Danvers, MA, USA), and α-tubulin (Santa-Cruz Biotechnology).
3
Cytokine Activation Pathway Analysis
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Phorbol-12-myristate-13-acetate (PMA), MSU, ATP, nigericin, poly A:T, Pam3CSK4, A9C, DIDS, and IAA94 were purchased from Sigma. MitoSox, MitoTracker Red, DAPI, MQAE, Lipofectamine RNAiMAX, Lipofectamine 2000, and M-MLV were from Invitrogen. Ultrapure LPS was obtained from Invivogen. SYRB Green was from TransGen Biotech. MnTBAP was from Santa Cruz. Salmonella is a gift from R.V. Bruggen. Anti-human cleaved IL-1β (2021, 1:1000 dilution) and anti-human caspase-1(2225, 1:1000 dilution) antibodies were purchased from Cell Signalling. Anti-human pro-IL-1β (60136-1-lg, 1:1000 dilution) anti-CLIC1(14545-1-AP, 1:500 dilution), anti-GAPDH (10494-1-AP, 1:1000 dilution), anti-VDAC1 (66345-1-lg, 1:1000 dilution), and anti-ATP1A1 (55187-1-AP, 1:1000 dilution) antibodies were from Proteintech. Anti-CLIC4 (SC-135739, 1:1000 dilution), anti-NEK7 (SC-50756, 1:500 dilution), and anti-ASC (rabbit, SC-22514, 1:400 dilution) antibodies were from Santa Cruz. Anti-ASC (mouse, 04–147, 1:1000 dilution) antibody was from Merck Millipore. Anti-CLIC5 (ACL-025, 1:250 dilution) antibody was from Alomone labs. Anti-mouse IL-1β (AF-401-NA, 1:1000 dilution) antibody was from R&D. Anti-mouse caspase-1 (p20) (AG-20B-0042, 1:1000 dilution) and anti-NLRP3 (AG-20B-0014, 1:1000 dilution) antibodies were from Adipogen. Anti-β-actin (P30002, 1:1000 dilution) antibody was from Abmart.
4
Metformin and IAA94 in Cell Characterization
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Metformin (1,1-dimethylbiguanide-hydrochloride) and IAA94 (indanyloxyacetic acid 94) were from Sigma-Aldrich (Milano, Italy).
Antibodies: anti-CLIC1, Santa Cruz (Dallas, USA); anti-GFAP, anti-Nestin, and anti-N cadherin, Abcam (Cambridge, UK); anti-α-tubulin, anti-β-III tubulin, anti-FLAG M2-Cy3 and anti-FLAG M2, Sigma-Aldrich (Milano, Italy); anti-Olig2 and anti-Sox2, Millipore (Vimodrone, Italy); anti-Rb, BD Biosciences (Milano, Italy); Alexa Fluor 488 anti-mouse and Alexa Fluor 546 anti-phalloidin, Invitrogen (Carlsbad CA, USA); DyLight 459-goat anti-rabbit IgG, Jackson Immunoresearch (West Grove, USA).
Antibodies: anti-CLIC1, Santa Cruz (Dallas, USA); anti-GFAP, anti-Nestin, and anti-N cadherin, Abcam (Cambridge, UK); anti-α-tubulin, anti-β-III tubulin, anti-FLAG M2-Cy3 and anti-FLAG M2, Sigma-Aldrich (Milano, Italy); anti-Olig2 and anti-Sox2, Millipore (Vimodrone, Italy); anti-Rb, BD Biosciences (Milano, Italy); Alexa Fluor 488 anti-mouse and Alexa Fluor 546 anti-phalloidin, Invitrogen (Carlsbad CA, USA); DyLight 459-goat anti-rabbit IgG, Jackson Immunoresearch (West Grove, USA).
5
Synthesis and Characterization of Cell Culture Reagents
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Metformin (1,1-dimethylbiguanide hydrochloride), phenformin [1-(2-phenylethyl)-biguanide hydrochloride], dissolved in water, and IAA94 (indanyloxyacetic acid 94), in ethanol, were from Sigma-Aldrich (Milan, Italy). Moroxydine, cycloguanil and proguanil, were synthesized according to the procedures previously reported (Curd and Rose, 1946 (link); Modest, 1956 (link); Shapiro et al., 1959 (link)) and stock solutions were prepared in DMSO. The correspondent amount of DMSO (for moroxydine, cycloguanil and proguanil, max 0.3%) or ethanol (for IAA94, max 0.2%) was added in the control points, although we observed that, at these concentrations, the vehicles did not modify any of the parameter tested. Growth factor-reduced MatrigelTM was from BD Biosciences (Franklin Lakes, NJ, United States), type 1 rat tail collagen from SERVA Electrophoresis GmbH (Heidelberg, Germany), and methylcellulose from Sigma-Aldrich.
The following antibodies were used: anti-CLIC1 cl. F9 and cl. 356.1 (Santa Cruz Biotechnology, Dallas, TX, United States) for immunofluorescence and Western blot experiments, respectively; anti-GFAP, anti-nestin, anti-Oct4, anti-Sox2, and anti-MAP-2 (Abcam, Cambridge, United Kingdom); anti-Olig2 and anti-Sox2, (Merck Vimodrone, Italy); anti-mouse and anti-rabbit Alexa Fluor 488 and 568 secondary antibodies (Thermo Fisher Scientific, Carlsbad, CA, United States).
The following antibodies were used: anti-CLIC1 cl. F9 and cl. 356.1 (Santa Cruz Biotechnology, Dallas, TX, United States) for immunofluorescence and Western blot experiments, respectively; anti-GFAP, anti-nestin, anti-Oct4, anti-Sox2, and anti-MAP-2 (Abcam, Cambridge, United Kingdom); anti-Olig2 and anti-Sox2, (Merck Vimodrone, Italy); anti-mouse and anti-rabbit Alexa Fluor 488 and 568 secondary antibodies (Thermo Fisher Scientific, Carlsbad, CA, United States).
6
Patch-Clamp Electrophysiology of CLIC1 Currents
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To measure CLIC 1-mediated current, the patch-clamp electrophysiology technique was performed. The cells were voltage-clamped in a perforated-patch whole-cell configuration as previously reported [15 (link)]. The voltage protocol consisted of 800 ms pulses from −80 mV to +80 mV (20 mV voltage steps). The holding potential was set according to the resting potential of the single cells. Analysis was performed using Clampfit 10.2 (Molecular Devices, Sunnyvale, CA, USA). CLIC 1-mediated chloride current was isolated from the other cell ionic currents by perfusing 100 μmol/L of the specific chloride channel inhibitor IAA94 (indanyloxyacetic acid 94, SigmaAldrich). The patch-clamp solutions used are as follows: bath (mmol/L): 125 NaCl, 5.5 KCl, 24 HEPES, 1 MgCl2, 0.5 CaCl2, 5 D-glucose, 10 NaOH, pH 7.4; pipette (mmol/L): 135 KCl, 10 HEPES, 10 NaCl, 1 MgCl2, pH 7.2.
7
Fibroblasts and SW620 Cells Treatment
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The fibroblasts were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Waltham, Massachusetts, USA, that was supplemented with 10% FBS (Sigma, St. Louis, Missouri, USA and penicillin-streptomycin (Sigma). The fibroblasts were treated with vehicle (DMSO, Sigma) or equal volumes of GANT61 (10 μM, Selleckchem, Houston Texas, USA, SIS3 (1–5 μM, Selleckchem), FH535 (10 μM, Tocris), NPPB (25 μM, Sigma), and IAA-94 (50 μM, Sigma) for 48 h at 37 °C and 5% CO2.
SW620 cells were obtained from ATCC. The cells were engineering by CRISPR/Cas9 to introduce a truncation in the PTCH1 coding sequence (PTCH1mut) using short guide RNA (sgRNA) targeting the region of interest (D1222fs) or an irrelevant scrambled sequence (WT PTCH1) and selected using puromycin for 72 h. The cells were sequence-verified. For the indicated treatments, the SW620 clones were incubated with GANT61 (20 µM) or SD208 (10 µM, Selleckchem) or equal volumes of DMSO for 24 h.
SW620 cells were obtained from ATCC. The cells were engineering by CRISPR/Cas9 to introduce a truncation in the PTCH1 coding sequence (PTCH1mut) using short guide RNA (sgRNA) targeting the region of interest (D1222fs) or an irrelevant scrambled sequence (WT PTCH1) and selected using puromycin for 72 h. The cells were sequence-verified. For the indicated treatments, the SW620 clones were incubated with GANT61 (20 µM) or SD208 (10 µM, Selleckchem) or equal volumes of DMSO for 24 h.
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