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Millipore pbs edta
Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as <t>PBS-EDTA,</t> to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
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Images

1) Product Images from "Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy"

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152112

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
Figure Legend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

Techniques Used: Concentration Assay, Buffer Exchange

(A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.
Figure Legend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

Techniques Used: Injection, Flow Cytometry, Size-exclusion Chromatography

(A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.
Figure Legend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

Techniques Used: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).
Figure Legend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

Techniques Used: Solubility, Centrifugation, Concentration Assay, Standard Deviation

2) Product Images from "Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy"

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152112

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
Figure Legend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

Techniques Used: Concentration Assay, Buffer Exchange

(A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.
Figure Legend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

Techniques Used: Injection, Flow Cytometry, Size-exclusion Chromatography

(A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.
Figure Legend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

Techniques Used: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).
Figure Legend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

Techniques Used: Solubility, Centrifugation, Concentration Assay, Standard Deviation

3) Product Images from "Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy"

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152112

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
Figure Legend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

Techniques Used: Concentration Assay, Buffer Exchange

(A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.
Figure Legend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

Techniques Used: Injection, Flow Cytometry, Size-exclusion Chromatography

(A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.
Figure Legend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

Techniques Used: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).
Figure Legend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

Techniques Used: Solubility, Centrifugation, Concentration Assay, Standard Deviation

4) Product Images from "Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy"

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152112

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
Figure Legend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

Techniques Used: Concentration Assay, Buffer Exchange

(A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.
Figure Legend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

Techniques Used: Injection, Flow Cytometry, Size-exclusion Chromatography

(A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.
Figure Legend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

Techniques Used: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).
Figure Legend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

Techniques Used: Solubility, Centrifugation, Concentration Assay, Standard Deviation

5) Product Images from "Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses"

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021703

Blood exposure of MSCs activates complement and effector cells. Lepirudin-anticoagulated blood was incubated with MSCs (black), and blood treated with either PBS (white) or 10mM EDTA (shaded) served as active or inactive control, respectively. ( A ) Plasma levels of C3a (ng/ml, n = 13), and ( B ) Plasma level of soluble C5b-9 complex (AU/ml, n = 13) were detected with ELISA. ( C ) Flow cytometric analysis of PBMCs and MSCs after labeling of individual blood aliquots with specific antibodies and subsequent erythrocyte lysis; the cells were first gated according their scatter profile (top panel, scatter plot) and representative histograms for triggering of CD11b-expression on PMNs (green) or binding of C3-fragments to MSCs (red) are shown compared to EDTA-inactivated negative control blood (grey histograms). ( D ) Up-regulation of CD11b on PMNs in blood (MFI, n = 19). The median fluorescence intensity (MFI) of the cell-surface marker CD11b was assessed with flow cytometry. ( E ) Percentage of recovered MSCs after a 40 min exposure to active or EDTA-inactivated blood (n = 17). The data in figure A-E are means±SEM; with: ns = not significant; * *P
Figure Legend Snippet: Blood exposure of MSCs activates complement and effector cells. Lepirudin-anticoagulated blood was incubated with MSCs (black), and blood treated with either PBS (white) or 10mM EDTA (shaded) served as active or inactive control, respectively. ( A ) Plasma levels of C3a (ng/ml, n = 13), and ( B ) Plasma level of soluble C5b-9 complex (AU/ml, n = 13) were detected with ELISA. ( C ) Flow cytometric analysis of PBMCs and MSCs after labeling of individual blood aliquots with specific antibodies and subsequent erythrocyte lysis; the cells were first gated according their scatter profile (top panel, scatter plot) and representative histograms for triggering of CD11b-expression on PMNs (green) or binding of C3-fragments to MSCs (red) are shown compared to EDTA-inactivated negative control blood (grey histograms). ( D ) Up-regulation of CD11b on PMNs in blood (MFI, n = 19). The median fluorescence intensity (MFI) of the cell-surface marker CD11b was assessed with flow cytometry. ( E ) Percentage of recovered MSCs after a 40 min exposure to active or EDTA-inactivated blood (n = 17). The data in figure A-E are means±SEM; with: ns = not significant; * *P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Labeling, Lysis, Expressing, Binding Assay, Negative Control, Fluorescence, Marker, Cytometry

6) Product Images from "Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy"

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0152112

Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
Figure Legend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

Techniques Used: Concentration Assay, Buffer Exchange

(A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.
Figure Legend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

Techniques Used: Injection, Flow Cytometry, Size-exclusion Chromatography

(A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.
Figure Legend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

Techniques Used: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).
Figure Legend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

Techniques Used: Solubility, Centrifugation, Concentration Assay, Standard Deviation

Related Articles

Diafiltration Assay:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. To remove DMSO and unreacted MS(PEG)4 in the AMB-PEG formulation, concentration and diafiltration with PBS-EDTA was performed using Amicon Ultra 0.5 mL 10 kDa centrifugal filters (Millipore, Billerica, MA), which retained AMB-PEG, at 4°C. .. Dynamic Light Scattering (DLS) analysis (described below) was subsequently carried out on diafiltered and non-diafiltered samples, and it was established that the diafiltration step did not significantly affect the particle size distribution of the AMB-PEG formulation.

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore). .. The AMB-PEG retentate was adjusted to a concentration of 2 mM, and subsequently serially diluted to obtain solutions ranging in concentrations from 0.05 to 2 mM.

Cytometry:

Article Title: Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation
Article Snippet: Paragraph title: Flow Cytometry ... Cells were centrifuged, washed one time in PBS/EDTA and incubated for 30 min at obscurity into 1ml containing 40μg/ml propidium iodide (Sigma), 200μg/ml RNAse A (Sigma), 0.1% triton X-100 (v/v, Sigma) in PBS EDTA.

Article Title: Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system
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Electrophoresis:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: Western blot analysis SDS-PAGE was performed in a Mini-Protean 3 electrophoresis apparatus according to the supplier's instructions (Bio-Rad, Hercules, CA). .. To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells.

Incubation:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: .. To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells. .. Proteins were solubilized with lysing buffer (1% SDS, 10 mM Tris-HCL pH 7.4, and protease inhibitors) and cell debris was pelleted at 13,000 g. Equal amounts of protein were separated on a 10% SDS-PAGE, electroblotted onto a PVDF membrane (Perkin-Elmer, Boston, MA), and probed with a 1:8000 dilution of peroxidase-labeled rabbit anti-human C3d-HRP and anti-C3c-HRP antibody (Dako, Glostrup, Denmark).

Article Title: Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation
Article Snippet: .. Cells were centrifuged, washed one time in PBS/EDTA and incubated for 30 min at obscurity into 1ml containing 40μg/ml propidium iodide (Sigma), 200μg/ml RNAse A (Sigma), 0.1% triton X-100 (v/v, Sigma) in PBS EDTA. ..

Article Title: Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation
Article Snippet: Once removed from the interphase and washed two to three times with PBS/EDTA, MNCs were suspended in a proliferation medium consisting of minimum essential medium alpha-modification (α-MEM, Sigma-Aldrich) containing 20% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich), and 2mM L-glutamine (Sigma-Aldrich). .. After 24 hours of incubation, non-adherent cells were removed and fresh medium was added to dishes as described in the literature [ ].

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation. ..

Activity Assay:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: To remove DMSO and unreacted MS(PEG)4 in the AMB-PEG formulation, concentration and diafiltration with PBS-EDTA was performed using Amicon Ultra 0.5 mL 10 kDa centrifugal filters (Millipore, Billerica, MA), which retained AMB-PEG, at 4°C. .. AMB-PEG formulations were then filtered using a 0.22 μm PVDF syringe filter (Millipore) prior to in vitro toxicity and activity tests.

Mass Spectrometry:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. To remove DMSO and unreacted MS(PEG)4 in the AMB-PEG formulation, concentration and diafiltration with PBS-EDTA was performed using Amicon Ultra 0.5 mL 10 kDa centrifugal filters (Millipore, Billerica, MA), which retained AMB-PEG, at 4°C. .. Dynamic Light Scattering (DLS) analysis (described below) was subsequently carried out on diafiltered and non-diafiltered samples, and it was established that the diafiltration step did not significantly affect the particle size distribution of the AMB-PEG formulation.

Western Blot:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: Paragraph title: Western blot analysis ... To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells.

Flow Cytometry:

Article Title: Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation
Article Snippet: Paragraph title: Flow Cytometry ... Cells were centrifuged, washed one time in PBS/EDTA and incubated for 30 min at obscurity into 1ml containing 40μg/ml propidium iodide (Sigma), 200μg/ml RNAse A (Sigma), 0.1% triton X-100 (v/v, Sigma) in PBS EDTA.

Article Title: Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system
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Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: Size exclusion chromatography (SEC) SEC was performed at a flow rate of 0.1 ml/min using a Superdex 75 PC 3.2/30 column (GE Healthcare, UK) equilibrated with the appropriate mobile phase. .. All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

Transfection:

Article Title: Human axial progenitors generate trunk neural crest cells in vitro
Article Snippet: In brief, a human BAC (RP11-12L16) with piggyBac transposon repeats flanking the bacterial backbone and with Venus inserted directly after the initiating methionine of MSGN1 was transfected together with a piggyBac Transposase into NCRM1 iPSCs. .. For NMP/axial progenitor differentiation hPSCs were dissociated using PBS/EDTA and plated at a density of 55,000 cells/cm2 (density optimised for 12-well plates) on fibronectin (Sigma) or vitronectin (Thermo Fisher)-coated wells, directly into NMP-inducing medium containing CHIR99021 (Tocris), FGF2 (20 ng/ml, R and D) and ROCK inhibitor Y-27632 (Tocris or Generon) for the first only day (10 µM, Tocris).

Transferring:

Article Title: Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system
Article Snippet: Cells were then dissociated in PBS/EDTA 0.5 mM for 2–3 min. PBS/EDTA was aspirated and replaced with 1 mL E8 medium containing 1.25 μM ROCK inhibitor (Y0503, Sigma). .. Cells were pipetted 3–4 times using a P1000 pipette to dissociate into small to medium sized clusters, and split onto new 6-well plates at various dilutions.

Cell Culture:

Article Title: Effects of Desferoxamine-induced Hypoxia on Neuronal Human mu-Opioid Receptor Gene Expression
Article Snippet: Paragraph title: Cell culture and counting ... Cells treated with deferoxamine (DFO) were rinsed gently with PBS, and detached with PBS/EDTA for cell counting using trypan blue staining (Sigma).

Article Title: Human axial progenitors generate trunk neural crest cells in vitro
Article Snippet: Paragraph title: Cell culture and differentiation ... For NMP/axial progenitor differentiation hPSCs were dissociated using PBS/EDTA and plated at a density of 55,000 cells/cm2 (density optimised for 12-well plates) on fibronectin (Sigma) or vitronectin (Thermo Fisher)-coated wells, directly into NMP-inducing medium containing CHIR99021 (Tocris), FGF2 (20 ng/ml, R and D) and ROCK inhibitor Y-27632 (Tocris or Generon) for the first only day (10 µM, Tocris).

Teratoma Formation Assay:

Article Title: Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system
Article Snippet: Pluripotency was confirmed by teratoma formation assay, G-banding karyotype and flow cytometry for TRA-1–60 and NANOG markers as previously described ( ). iPSCs were maintained on 6-well tissue culture plates thinly coated with Matrigel® (Corning, 354230) in Essential 8™ (E8) Medium (A1517001, Thermo-Fisher). .. Cells were then dissociated in PBS/EDTA 0.5 mM for 2–3 min. PBS/EDTA was aspirated and replaced with 1 mL E8 medium containing 1.25 μM ROCK inhibitor (Y0503, Sigma).

Inhibition:

Article Title: Human axial progenitors generate trunk neural crest cells in vitro
Article Snippet: For NMP/axial progenitor differentiation hPSCs were dissociated using PBS/EDTA and plated at a density of 55,000 cells/cm2 (density optimised for 12-well plates) on fibronectin (Sigma) or vitronectin (Thermo Fisher)-coated wells, directly into NMP-inducing medium containing CHIR99021 (Tocris), FGF2 (20 ng/ml, R and D) and ROCK inhibitor Y-27632 (Tocris or Generon) for the first only day (10 µM, Tocris). .. BMP inhibition was carried out using LDN193189 (Tocris) at 100 nM.

Sonication:

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: DNA were sonicated on Q Sonica Sonicator Q700 for 8 min (30 sec ON, 30 s OFF) to generate fragments of 200–500 bp. .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation.

Injection:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection. .. MALDI-TOF Analysis The MALDI-TOF data was acquired on a 5800 MALDI-TOF/TOF mass spectrometer (AB Sciex, Foster City, CA) in positive reflectron mode.

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: For SEC performed using PBS-EDTA as the mobile phase, 3 μL of 2 mM AMB-PEG conjugate formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analyzed. .. All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

Magnetic Beads:

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation. ..

Isolation:

Article Title: Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation
Article Snippet: Paragraph title: Cord blood collection, and isolation and culture of hMSC ... Once removed from the interphase and washed two to three times with PBS/EDTA, MNCs were suspended in a proliferation medium consisting of minimum essential medium alpha-modification (α-MEM, Sigma-Aldrich) containing 20% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich), and 2mM L-glutamine (Sigma-Aldrich).

Immunodetection:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells. .. Purified C3b, iC3b, and C3d (1 µg/lane) were used as positive controls for immunodetection.

Purification:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells. .. Purified C3b, iC3b, and C3d (1 µg/lane) were used as positive controls for immunodetection.

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: For DRIP, the chromatin preps were treated with 20 mg/mL Proteinase K (Thermo Fisher Scientific) at 65 °C overnight and total DNA was purified by phenol/chloroform purification method. .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation.

FACS:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: Cells were prepared as described above (NHS or NHS/EDTA in FACS tubes for 20 min at 37°C). .. To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells.

Lysis:

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: Cells were cross-linked in 1% formaldehyde for 10 min before quenching with glycine for 5 min at room temperature, and then lysed in ChIP lysis buffer (50 mm HEPES-KOH at pH 7.5, 140 mm NaCl, 1 mm EDTA at pH 8, 1% Triton X-100, 0.1% Na-Deoxycholate, 1% SDS) and rotated for 1 h at 4 °C. .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation.

Chromatin Immunoprecipitation:

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: Paragraph title: DRIP and ChIP-qPCR ... Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation.

SDS Page:

Article Title: Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses
Article Snippet: Western blot analysis SDS-PAGE was performed in a Mini-Protean 3 electrophoresis apparatus according to the supplier's instructions (Bio-Rad, Hercules, CA). .. To remove unbound proteins, cell pellets were washed three times with 10 mM PBS/EDTA, resuspended with protease inhibitors (SigmaFast; Sigma-Aldrich Sweden AB) and incubated with 0.1 M methylamine (pH 9.0) for 1 h at 37°C, to disrupt the covalent linkage of C3 fragments to the cells.

Software:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore). .. These values and the temperature at which they were acquired were entered into the particle sizing software for analysis.

In Vitro:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: To remove DMSO and unreacted MS(PEG)4 in the AMB-PEG formulation, concentration and diafiltration with PBS-EDTA was performed using Amicon Ultra 0.5 mL 10 kDa centrifugal filters (Millipore, Billerica, MA), which retained AMB-PEG, at 4°C. .. AMB-PEG formulations were then filtered using a 0.22 μm PVDF syringe filter (Millipore) prior to in vitro toxicity and activity tests.

Size-exclusion Chromatography:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: Paragraph title: Size exclusion chromatography (SEC) ... All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

Article Title: MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts
Article Snippet: DNA were sonicated on Q Sonica Sonicator Q700 for 8 min (30 sec ON, 30 s OFF) to generate fragments of 200–500 bp. .. Protein A magnetic beads (Bio-rad) were first pre-blocked with PBS/EDTA containing 0.5% BSA and then incubated with S9.6 antibody (1:200, clone S9.6, MABE1095, Millipore) in IP buffer (50 mm Hepes/KOH at pH 7.5; 0.14 m NaCl; 5 mm EDTA; 1% Triton X-100; 0.1% Na-Deoxycholate, ddH2 O) at 4 °C for 4 h with rotation.

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. For SEC performed using PBS-EDTA as the mobile phase, 3 μL of 2 mM AMB-PEG conjugate formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analyzed. .. All samples dispersed in PBS-EDTA were 0.22 μm-filtered (Millipore) prior to column injection.

Concentration Assay:

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: .. To remove DMSO and unreacted MS(PEG)4 in the AMB-PEG formulation, concentration and diafiltration with PBS-EDTA was performed using Amicon Ultra 0.5 mL 10 kDa centrifugal filters (Millipore, Billerica, MA), which retained AMB-PEG, at 4°C. .. Dynamic Light Scattering (DLS) analysis (described below) was subsequently carried out on diafiltered and non-diafiltered samples, and it was established that the diafiltration step did not significantly affect the particle size distribution of the AMB-PEG formulation.

Article Title: Human axial progenitors generate trunk neural crest cells in vitro
Article Snippet: For NMP/axial progenitor differentiation hPSCs were dissociated using PBS/EDTA and plated at a density of 55,000 cells/cm2 (density optimised for 12-well plates) on fibronectin (Sigma) or vitronectin (Thermo Fisher)-coated wells, directly into NMP-inducing medium containing CHIR99021 (Tocris), FGF2 (20 ng/ml, R and D) and ROCK inhibitor Y-27632 (Tocris or Generon) for the first only day (10 µM, Tocris). .. We observed some variation in terms of induction of T + SOX2+NMPs both between hPSC lines and also batches of CHIR99021 and thus the concentration of the latter was varied between 3–4 µM.

Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy
Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore). .. The AMB-PEG retentate was adjusted to a concentration of 2 mM, and subsequently serially diluted to obtain solutions ranging in concentrations from 0.05 to 2 mM.

Cell Counting:

Article Title: Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation
Article Snippet: Once removed from the interphase and washed two to three times with PBS/EDTA, MNCs were suspended in a proliferation medium consisting of minimum essential medium alpha-modification (α-MEM, Sigma-Aldrich) containing 20% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich), and 2mM L-glutamine (Sigma-Aldrich). .. Cell counting was performed using an automated cell analyzer (Coulter Counter Multisizer 4, Beckman Coulter).

Article Title: Effects of Desferoxamine-induced Hypoxia on Neuronal Human mu-Opioid Receptor Gene Expression
Article Snippet: .. Cells treated with deferoxamine (DFO) were rinsed gently with PBS, and detached with PBS/EDTA for cell counting using trypan blue staining (Sigma). .. Cellular glutathione level was determined using GSH-Glo Glutathione Assay (Promega).

BAC Assay:

Article Title: Human axial progenitors generate trunk neural crest cells in vitro
Article Snippet: In brief, a human BAC (RP11-12L16) with piggyBac transposon repeats flanking the bacterial backbone and with Venus inserted directly after the initiating methionine of MSGN1 was transfected together with a piggyBac Transposase into NCRM1 iPSCs. .. For NMP/axial progenitor differentiation hPSCs were dissociated using PBS/EDTA and plated at a density of 55,000 cells/cm2 (density optimised for 12-well plates) on fibronectin (Sigma) or vitronectin (Thermo Fisher)-coated wells, directly into NMP-inducing medium containing CHIR99021 (Tocris), FGF2 (20 ng/ml, R and D) and ROCK inhibitor Y-27632 (Tocris or Generon) for the first only day (10 µM, Tocris).

Staining:

Article Title: Effects of Desferoxamine-induced Hypoxia on Neuronal Human mu-Opioid Receptor Gene Expression
Article Snippet: .. Cells treated with deferoxamine (DFO) were rinsed gently with PBS, and detached with PBS/EDTA for cell counting using trypan blue staining (Sigma). .. Cellular glutathione level was determined using GSH-Glo Glutathione Assay (Promega).

Gradient Centrifugation:

Article Title: Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation
Article Snippet: After 1:1 blood dilution with phosphate-buffered saline (PBS)/2 mM EDTA, mononuclear cells (MNCs) were isolated by density gradient centrifugation at 400 g and 20°C for 40 minutes using a Ficoll-Paque PLUS (GE Healthcare). .. Once removed from the interphase and washed two to three times with PBS/EDTA, MNCs were suspended in a proliferation medium consisting of minimum essential medium alpha-modification (α-MEM, Sigma-Aldrich) containing 20% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich), and 2mM L-glutamine (Sigma-Aldrich).

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    Millipore pbs edta
    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as <t>PBS-EDTA,</t> to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.
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    Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Representative absorbance spectra of AMB-PEG 2 and unconjugated AMB formulations prepared in buffers with varying hydrophobicity. 20 mM AMB-PEG 2 and unconjugated AMB formulations were prepared in DMSO and resuspended in 20% and 48% ACN buffers containing 4.3% acetic acid, as well as PBS-EDTA, to a final concentration of 2 mM AMB. As buffer hydrophobicity increases with higher ACN concentrations, the A 348 /A 409 ratio decreases, implying that AMB-PEG is increasingly in its monomeric form. As AMB-PEG 1 and 2 have similar UV-visible absorption profiles, with identical peak height ratios in all buffers tested, data for AMB-PEG 1 is not shown. AMB-PEG formulations that have been subjected to buffer exchange to PBS-EDTA through a 10 kDa centrifugal filter have the same UV-visible absorption spectra as the initial formulation of AMB-PEG in PBS-EDTA, which contains 10% DMSO.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Concentration Assay, Buffer Exchange

    (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) Reverse phase chromatogram of AMB-PEG and unconjugated AMB, with eluted peaks detected at 406 nm. 50 μL of AMB-PEG 1 and 2, and 10 μL of unconjugated AMB dispersed in PBS-EDTA and 48% ACN respectively were injected into a C18 reverse phase column and eluted isocratically in a 48% ACN buffer at a flow rate of 0.5 ml/min for 40 minutes. Peaks were detected at 406 nm. The AMB-PEG conjugate has a shorter retention time, implying that it is more hydrophilic. From the AMB-PEG samples, AMB-PEG conjugate and free AMB (based on the retention time of unconjugated AMB) fractions were collected for further analysis via size exclusion chromatography. (B) Size exclusion chromatogram of AMB-PEG 2 and unconjugated AMB, as well as the relevant fractions collected from RPC, in a 20% ACN mobile phase. 20 mM of AMB-PEG 2 formulations and unconjugated AMB in DMSO were prepared and resuspended at 2 mM in a 20% ACN buffer. 50 μL of these samples (unconjugated AMB diluted tenfold prior to analysis) and the peak fractions collected previously from RPC were passed through a size exclusion column and eluted peaks were detected at 406 nm. Unconjugated AMB was eluted later compared to the AMB-PEG conjugate, implying that it has a smaller hydrodynamic volume compared to AMB-PEG in 20% ACN. The previously collected AMB-PEG conjugate and free AMB peak fractions had similar retention times as the AMB-PEG 2 formulation and unconjugated AMB samples respectively, thus verifying their respective peak identities. AMB-PEG 1 and 2 have identical elution profiles. (C) Size exclusion chromatogram of AMB-PEG and unconjugated AMB dispersed in PBS-EDTA. 3 μL of 2 mM AMB-PEG formulations that have been retained by 10 kDa centrifugal filters (Millipore) and 50 μL of the supernatant obtained from unconjugated AMB were analysed using a Superdex 75 size exclusion column and eluted peaks detected at 406 nm. In a PBS-EDTA mobile phase, unconjugated AMB has a shorter retention time of 20 minutes compared to AMB-PEG at 40 minutes, implying that AMB-PEG has a smaller hydrodynamic volume under these experimental conditions.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Injection, Flow Cytometry, Size-exclusion Chromatography

    (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: (A) AMB-PEG reaction scheme. The NHS ester on MS(PEG) 4 reacts with the primary amine (–NH2) group on AMB at a 1:1 molar ratio, generating conjugated AMB-PEG via amide bond formation. (B) Solubility of AMB-PEG at varying AMB:PEG molar ratios. AMB-PEG mixtures and unreacted AMB were prepared in DMSO, incubated for 2 hours and then (i) dispersed to a final concentration of 2 mM in PBS-EDTA, containing 10% DMSO by volume. (ii) The mixtures were then centrifuged to facilitate the observation of insoluble precipitates. Higher molar ratios yielded a suspension of yellow particles that precipitated upon centrifugation, similar to what was observed with unconjugated AMB.

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Mass Spectrometry, Solubility, Incubation, Concentration Assay, Centrifugation

    Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Journal: PLoS ONE

    Article Title: Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy

    doi: 10.1371/journal.pone.0152112

    Figure Lengend Snippet: Aqueous solubility of AMB-PEG 1 and 2. Increasing amounts of AMB-PEG formulations were added to PBS-EDTA, and post-centrifugation, the concentration of soluble AMB-PEG in the supernatants quantified based on their absorbance at 365 nm. Error bars denote the standard deviation between 3 independent formulations. Dotted line represents the theoretical condition where AMB formulation is completely soluble (i.e. concentration of total AMB = concentration of soluble AMB).

    Article Snippet: AMB-PEG Particle Size Determination Using DLS 2 mM solutions of AMB-PEG were prepared in PBS-EDTA, and diafiltration carried out with PBS-EDTA using 10 kDa centrifugal filters (Millipore).

    Techniques: Solubility, Centrifugation, Concentration Assay, Standard Deviation