Ivacaftor and colistin were procured from AOK Chem (Shanghai, China) and Betapharma Co. Ltd. (Jiangsu, China), respectively. Bovine serum albumin (BSA, purity ＞ 98%, Mw 66.5 kDa), glutaraldehyde, L-leucine, Neutral Red and Alamar Blue dye were obtained from Sigma-Aldrich Inc. (St. Louis, MO, USA). MTT assay kits and DMSO were obtained from Thermo Fisher scientific Inc. (Waltham, MA, USA). Hoechst 33342 was provided by Becton Dickinson (Franklin Lakes, NJ, USA). The fetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM) and Hanks’ Balanced Salt Solution (HBSS) were purchased from Gibco Life Technologies Corporation (Eugene, OR, USA). BCA protein assay kit, TritonX-100 and RIPA buffer were obtained from Thermo Scientific (Rockford, IL, USA). Transwell permeable membrane cell culture inserts (pore diameter 0.4 μm, insert diameter 6.5 mm) were purchased from Corning Incorporated (Kennebunk, ME, USA). Paraformaldehyde (PFA) was purchased from Biotium, Inc. (Fremont, CA, USA). Mouse anti-polymyxin B IgM, Goat anti-mouse IgM antibody conjugated with Alexa Fluor™ 647, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Invitrogen (Life Technologies Corporation, Eugene, OR, USA).
Alamar blue dye
Manufactured by Merck Group
Sourced in United States, France
Alamar Blue dye is a laboratory reagent used to measure cellular metabolic activity and viability. It is a non-toxic, water-soluble dye that changes color in response to chemical reduction, indicating the metabolic state of cells. The dye can be used with a variety of cell types and is commonly employed in cell proliferation, cytotoxicity, and viability assays.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Mtt assay kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Neutral red, by Merck Group (1 mentions)
Hoechst 33342, by BD (1 mentions)
L leucine, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Aria 3 flow cytometer, by BD (1 mentions)
Pi rnase staining solution, by Thermo Fisher Scientific (1 mentions)
Annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Rifampicin rif, by Merck Group (1 mentions)
Tween 40, by Merck Group (1 mentions)
Dtx880, by Beckman Coulter (1 mentions)
4 protocols using alamar blue dye
1
Antimicrobial Efficacy Evaluation
2
Evaluating Cell Viability and Apoptosis
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Cell viability was measured by cell count using trypan blue dye (Sigma, T8154) /hemocytometer or resazurin (Alamarblue dye, Sigma) with the Envision Fluorescent Reader (Perkin Elmer). Apoptosis was assessed using Annexin-V/Dead Cell Apoptosis Kit (V13241, ThermoFisher) and cell cycle analysis with PI/RNase Staining solution (F10797, ThermoFisher) as per manufactures’ instructions. Data were acquired using an LSRII flow cytometer and an Aria III flow cytometer (BD Biosciences, UK) and analyzed using FlowJo software (Tree Star Inc., USA).
3
Rifampicin Stock Preparation and Storage
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Rifampicin (Rif) were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Stock solutions at 1000 μg/mL were filter sterilized and stored at -20°C. Working solutions were prepared at four times the final higher concentration in Middlebrook 7H9 supplemented with 2% glycerol, 10% OADC (oleic acid, albumin, dextrose, and catalase) and 0.05% (vol/vol) Tween 40 (Sigma). Alamar blue dye 0.01% (weight/volume) from Sigma-Aldrich (St. Quentin Fallavier, France) was prepared and sterilized by filtration, and stored at 4°C for up to 1 week. Kit SGM Italia were also obtained from Sigma-Aldrich.
4
Alamar Blue Assay for Cell Viability
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Cell viability was quantified using the Alamar Blue assay, as described previously by Ahmed et al. (1994) with minor modifications (Rodrigues et al. 2015) . Non-tumor and tumor cells were placed in 96-well plates (7 x 10 4 cells/mL for adherent cells or 3 x 10 5 cells/mL for suspended cells in 100 µL of medium) and incubated with EEGP or its fractions (0.39-50 µg/mL) for 72 h at 37°C under a CO 2 (5%) atmosphere. Cell viability was quantified based on the ability of living cells to reduce Alamar Blue dye (52 µg/mL; Sigma Aldrich) to a red resorufin product where the absorbance was measured at 570 and 600 nm (DTX-880, Beckman Coulter) after 2h at 37°C under CO 2 (5%) atmosphere. Doxorubicin (purity 95%, doxorubicin hydrochloride, Laboratory IMA) was used as the positive control (0.08-5 µg/mL).
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