Triptolide (molecular weight 360.4, purity ≥98% by HPLC), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and protease inhibitor solution were obtained from Sigma Chemical (St. Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, LipofectAMINE 2000 and carboxyfluorescein diacetate succinimidyl ester were purchased from Invitrogen (Camarillo, CA, USA). The cytokine and chemokine ELISA assay kits were obtained from R&D Systems (Minneapolis, MN, USA). RIPA was supplied by Thermo Scientific (Waltham, MA, USA). A protein assay kit and polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad Laboratories (Hercules, CA, USA).
Carboxyfluorescein diacetate succinimidyl ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
Carboxyfluorescein diacetate succinimidyl ester is a fluorescent dye used for labeling proteins and peptides. It is a cell-permeant compound that can be used to monitor cell viability and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (9 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
96 well polystyrene plate, by SPL Life Sciences (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Nis elements c version 3, by Nikon (2 mentions)
Iris digital cell imaging system, by Logos Biosystems (2 mentions)
Facscalibur, by BD (2 mentions)
Protease inhibitor solution, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Triptolide, by Merck Group (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Bio-Rad (1 mentions)
Tiron, by Merck Group (1 mentions)
8 isoprostane assay kit, by Cayman Chemical (1 mentions)
44 protocols using carboxyfluorescein diacetate succinimidyl ester
1
Triptolide Modulates Inflammatory Response
2
Ox-LDL-induced Oxidative Stress Assay
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TP (molecular weight = 360.4 and purity ≥98% by HPLC, as described in the product sheet supplied by Sigma-Aldrich (St Louis, Missouri), catalog No. T3652), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), proteinase inhibitor solution, dimethyl sulfoxide (DMSO), Tiron, and Triton X-100 were purchased from Sigma-Aldrich. Ox-LDL was purchased from the Beijing Solarbio Life Science Company (Beijing, China). RPMI-1640 medium, carboxyfluorescein diacetate succinimidyl ester, fetal bovine serum, and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, California). Cytokine and chemokine ELISA kits were obtained from R&D Systems (Minneapolis, Minnesota). An 8-isoprostane assay kit was purchased from Cayman Chemical (Ann Arbor, Michigan). Malondialdehyde (MDA) and superoxide dismutase (SOD) activity assay kits were obtained from Beyotime Biotechnology Institute (Shanghai, China). A protein assay kit and RIPA lysis buffer were supplied by Bio-Rad Laboratories (Hercules, California) and Thermo Scientific (Waltham, Massachusetts), respectively.
3
Quantifying Candida albicans Biofilm Formation
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Candida albicans biofilms were grown in 96‐well plates in the absence or presence of 7‐benzyloxyindole (0.1 mM) without shaking. Planktonic cells were then removed by washing with H2O three times. Carboxyfluorescein diacetate succinimidyl ester (a minimally fluorescent lipophile; Catalog #: C34554; Invitrogen, Molecular Probes, Inc, Eugene, OR, USA)(Weston and Parish,1990 ) was used to stain C. albicans cells. The bottom of 96‐well plates was visualized using an (a 488 nm) Ar laser (emission wavelength 500 to 550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo, Japan). Colour confocal images were constructed using NIS‐Elements C version 3.2 (Nikon Eclipse), and images were obtained with a 20× objective (Kim et al., 2012 ). For each experiment, two independent cultures were examined for at least 10 random positions. Biofilm formation was quantified by converting colour confocal images (20 image stacks) to grey scale using ImageJ, and COMSTAT biofilm software (Heydorn et al., 2000 ) was used to calculate biomasses (μm3 μm−2), mean biofilm thicknesses (μm) and substratum coverages (%). For each biofilm image, stack threshold was fixed and divided into four positions and 20 planar images per position were analysed.
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Candida albicans biofilms were grown in 96‐well plates in the absence or presence of 7‐benzyloxyindole (0.1 mM) without shaking. Planktonic cells were then removed by washing with H2O three times. Carboxyfluorescein diacetate succinimidyl ester (a minimally fluorescent lipophile; Catalog #: C34554; Invitrogen, Molecular Probes, Inc, Eugene, OR, USA)(Weston and Parish,
4
Quantification of V. parahaemolyticus Biofilm
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The inoculum of V. parahaemolyticus was grown in 96-well polystyrene plates (SPL life sciences, Pocheon-si, South Korea) without shaking at 30 °C for 24 h in the presence or absence of CNMA and its selected derivatives (50 µg/mL). The formed biofilms were stained with 100 μL of pre-warmed PBS containing CFSE (carboxyfluorescein diacetate succinimidyl ester) (Invitrogen, Eugene, OR, USA) for 20 min at 37 °C and were visualized by CLSM (Nikon Eclipse Ti, Nikon Instruments, Tokyo, Japan), as described by [37 (link)]. At least 10 random positions in each of the three independent cultures were chosen for microscopic analysis [38 (link)]. Biofilm formation was quantified using COMSTAT biofilm software [39 (link)] to determine the biomasses (µm3/µm2), mean thicknesses (µm), and substratum coverage (%).
5
Quantifying Bacterial Biofilm Formation
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Escherichia coli BW25113 and S. aureus cells were cultured in 96-well polystyrene plates (SPL Life Sciences, Korea) without shaking with or without 5-iodoindole. Biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) (Weston and Parish 1990 (link)). Planktonic cells were removed by washing with PBS three times, and static biofilms were visualized by excitation using an Ar laser 488 nm (emission wavelengths 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo) using a 20× objective (Kim et al. 2012 (link)). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were chosen for microscopic analysis.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software (Heydorn et al. 2000 (link)) was used to determine biomasses (μm3 μm−2), mean thicknesses (μm), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.
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Escherichia coli BW25113 and S. aureus cells were cultured in 96-well polystyrene plates (SPL Life Sciences, Korea) without shaking with or without 5-iodoindole. Biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) (Weston and Parish 1990 (link)). Planktonic cells were removed by washing with PBS three times, and static biofilms were visualized by excitation using an Ar laser 488 nm (emission wavelengths 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo) using a 20× objective (Kim et al. 2012 (link)). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were chosen for microscopic analysis.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software (Heydorn et al. 2000 (link)) was used to determine biomasses (μm3 μm−2), mean thicknesses (μm), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.
6
Quantifying Candida albicans Biofilms
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Candida albicans biofilms were produced on 96-well polystyrene plates in the presence or absence of α-methyl, trans-4-methyl, or trans-cinnamaldehydes at 50 μg/mL without shaking for 24 h at 37°C. After incubation, planktonic cells were removed by washing (three times) with distilled water, and biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Eugene, OR, United States) (Lee et al., 2019a (link)). Plate bottoms were then visualized using a 488 nm Ar laser (emission 500–550 nm) beneath a confocal laser microscope (Nikon Eclipse Ti, Tokyo, Japan). To quantify biofilm structures, COMSTAT software (Heydorn et al., 2000 (link)) was used to determine biovolumes (μm3 μm–2), mean biofilm thicknesses (μm), and percentage substratum coverages (%). Two autonomous cultures were performed for each experimental condition and at least 10 random positions were screened.
7
Evaluating MDSC's Immunosuppressive Activity
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An in vitro T-cell assay was performed as described to evaluate the MDSC’s activity [22 (link)]. Briefly, 1 × 105 carboxy fluorescein diacetate succinimidyl ester (Invitrogen, California, USA) labeled T-cells were activated with a 1/800 dilution of anti-CD3/CD28 antibody coated beads (Invitrogen, California, USA) and cultured with or without 5 × 104 WT or KO MDSCs. When indicated, the GzmB inhibitor, Z-AAD-CMK (Enzo Life Sciences, Antwerpen, Belgium), was added (10 μM). T-cell proliferation and viability were determined in flow cytometry.
8
Quantifying Candida albicans Biofilm Growth
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C. albicans biofilms were grown on 96-well plates with or without 6-gingerol or 6-shogaol without shaking for 24 h. Planktonic cells were then removed by washing with water three times, and biofilms were stained with carboxyfluorescein diacetate succinimidyl ester (a minimally fluorescent lipophile; Invitrogen, Molecular Probes, Inc, Eugene, USA) (Lee et al., 2016 (link)). Plate bases were then visualized using an (a 488 nm) Ar laser (emission 500 to 550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo), and COMSTAT biofilm software (Heydorn et al., 2000 (link)) was then used to calculate biovolumes (μm3 μm−2), mean biofilm thicknesses (μm), and substratum coverages (%). Two independent cultures were performed under each experimental condition and at least 10 random positions were assayed.
9
Quantifying Polymicrobial Biofilm Inhibition
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Polymicrobial biofilms were formed in 96-well polystyrene plates as described in section “Antibiofilm Activities of Indole Derivatives Against Polymicrobial Biofilms” with or without indole derivatives at a concentration of 100 μg/ml. To visualize biofilms, wells were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc., Eugene, United States). Biofilm structures were evaluated by visualizing plate bases using a 488-nm excitation Ar laser (emission 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo, Japan), and their spatial characteristics were quantified using the MATLAB R2007b program by analyzing at least four random positions in three independent cultures. To measure biofilm formation, color confocal images (20 image stacks) were converted to greyscale using ImageJ. MATLAB R2007b biofilm software was used to determine biomasses (μm3 per μm2), mean thicknesses (μm), and substratum coverages (%).
10
Suppression Assay for Allograft-specific Tregs
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For the suppression assay, CD4+CD25+ Tregs were isolated from the spleens of the allograft recipients that had been treated with anti-CD154 or isotype IgG 14 days following transplantation. CD4+CD25− T cells were isolated from the spleens of naïve BALB/c mice. The isolation was conducted using a CD4+CD25+ Regulatory T cell Isolation kit, according to the manufacturer’s instruction (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the prepared cells was >95%, as determined by flow cytometry. Fresh isolated CD4+CD25− T cells were labeled with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (Invitrogen Life Technologies, Carlsbad, CA, USA) and served as T effector (Teff) cells. After washing, 1×105 Teff cells were co-cultured in triplicate with 2×105 mitomycin-treated C57 BL/6 splenocytes in the presence or absence of Tregs for 72 h. The ratio of Tregs to Teff cells varied from 1:1 to 1:8. The alloantigen-stimulated Teff cell proliferation was determined by flow cytometry and suppression was calculated using the following formula: Suppression (%) = (Teff proliferation without Tregs - Teff proliferation with Tregs)/(Teff proliferation without Tregs) × 100.
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