T84 (#CCL-248) and Caco2 (#HTB-37) human epithelial cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA) and were cultured in 95% air with 5% CO2 at 37°C according to instructions provided by ATCC. Where indicated, cells were cultured on 5.0 μM or 0.4 μm pore polyester transwell inserts for 10–14 days to obtain confluent cell monolayers as measured by transepithelial resistance (CoStar, Cambridge, MA). Bumetanide (10 μM), S3226 (5 μM), indole (1 mM), myricetin (10 μM), and 4-aminobenzoic hydrazide (4-ABAH) (500 μM) obtained from Sigma–Aldrich (St. Louis, MA) were added to sterile filtered HBSS (indole and 4-ABAH) or DMSO (Bumetanide and S3226). Anti-CD11b (M1/70) and IgG1 rat isotype control monoclonal antibodies (10 μg/ml) were obtained from BioLegend (San Diego, CA). Anti-CD47 (B6H12) and IgG1 mouse isotype control monoclonal antibody (10 μg/ml) was obtained from Fisher Scientific (Hampton, NH). Anti-CD55 (clone OE-1) mouse monoclonal antibody (10 μg/ml) was produced as previously described.14 (link) All inhibitors were used at established, noncytotoxic concentrations.
S3226
Manufactured by Merck Group
The S3226 is a laboratory equipment manufactured by Merck Group. It is designed for general laboratory applications. The core function of the S3226 is to perform precise measurements and analyses in a controlled laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
4 4 diisothiocyanatostilbene 2 2 disulfonate, by Merck Group (1 mentions)
Cftrinh 172, by Cayman Chemical (1 mentions)
Bumetanide, by Merck Group (1 mentions)
Indole, by Merck Group (1 mentions)
4 abah, by Merck Group (1 mentions)
Myricetin, by Merck Group (1 mentions)
Caco 2 htb 37, by American Type Culture Collection (1 mentions)
T 84 ccl 248, by American Type Culture Collection (1 mentions)
Aphidicolin, by Abcam (1 mentions)
Verteporfin, by Bio-Techne (1 mentions)
Cytochalasin a, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
3 protocols using s3226
1
Epithelial Cell Culture Protocol
2
Pharmacological Inhibitors of Anion Transporters
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CFTRinh-172 was purchased from Cayman Chemical (Ann Arbor, MI), 4,4’-Diisothiocyanatostilbene-2,2’-disulfonate (DIDS) and S3226 were purchased from Sigma-Aldrich (St. Louis, MO). DRAinh-A250 and DRAinh-A270 were gifts from Drs. Alan Verkman and Onur Cil at the University of California, San Francisco. Linaclotide was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).
3
Preparation of Hypertonic and Hypotonic Solutions
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Sucrose(Macklin, S818046) was dissolved in ddH 2 O, the concentration of the stock solution was 1 mol/L. The stock solution is diluted by KSOM (260 mOsm) into a hypertonic solution(340 mOsm) of sucrose with a final concentration of 100 mM. We used ddH 2 O to dilute KSOM into a 70% hypotonic solution (170 mOsm). (-)-Blebbistatin (Sigma,B0560) was dissolved in DMSO at a concentration of 25 mM. The stock solution was diluted by KSOM to a working concentration of 1 μM. Dissolve LPA(Cayman,62215) in ethonal at the stock concentration of 1 mM. The stock solution was diluted by KSOM to a working concentration of 5 μM. Cytochalasin A(Sigma,C6637) was dissolved in DMSO at the stock concentration of 0.5 mg/mL. The stock solution was diluted by KSOM to a working concentration of 1 μg/mL. Dissolved S3226 (Sigma,SML 1996) in ddH 2 O to prepare a stock solution with a concentration of 2 mg/mL. The stock solution was diluted by KSOM to a working concentration of 20 nM. Dissolved Verteporfin(Tocris,5305) in DMSO to prepare a stock solution with a concentration of 10 mg/mL. The stock solution was diluted by KSOM to a working concentration of 2 μg/mL. Dissolved Aphidicolin (Abcam,ab142400) in DMSO to prepare a stock solution with a concentration of 10 mg/mL. The stock solution was diluted by KSOM to a working concentration of 2 μg/mL.
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