Journal: Journal of Biomedicine and Biotechnology

Article Title: Meso Scale Discovery and Luminex Comparative Analysis of Calbindin D28K

doi: 10.1155/2009/187426

Figure Lengend Snippet: (a) Calbindin D28K standard curve analysis on MSD. (b) Luminex analysis of calbindin D28K; with standard curve indicating the fluorescence intensity (FI) at the different concentrations.

Article Snippet: Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms.

Techniques: Luminex, Fluorescence

Journal: Journal of Biomedicine and Biotechnology

Article Title: Meso Scale Discovery and Luminex Comparative Analysis of Calbindin D28K

doi: 10.1155/2009/187426

Figure Lengend Snippet: Linearity analysis of rat cortical kidney homgenate dilutions.

Article Snippet: Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms.

Techniques: Luminex

Journal: Journal of Biomedicine and Biotechnology

Article Title: Meso Scale Discovery and Luminex Comparative Analysis of Calbindin D28K

doi: 10.1155/2009/187426

Figure Lengend Snippet: Precision analysis using rat cortical kidney homogenate.

Article Snippet: Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms.

Techniques: Luminex

Journal: Journal of Biomedicine and Biotechnology

Article Title: Meso Scale Discovery and Luminex Comparative Analysis of Calbindin D28K

doi: 10.1155/2009/187426

Figure Lengend Snippet: Interassay variation analysis of rat cortical kidney homogenate.

Article Snippet: Comparative analysis of the immunoassay was performed on the Meso Scale Development (MSD) and Luminex platforms.

Techniques: Luminex

Journal: Advanced Science

Article Title: Molecular Determinants of Protein Pathogenicity at the Single‐Aggregate Level

doi: 10.1002/advs.202410229

Figure Lengend Snippet: Aβ40 and Aβ42 uptake and subsequent cytokine secretion by iMGLs A) Schematic of the assessing Aβ uptake by iMGLs and measuring cytokine levels (IL‐1β, IL‐6, TNF‐α) in the media using ELISA. B,C) Representative images of iMGLs (stained with DAPI/Iba1) after uptake of B) Aβ42 and C) Aβ40 aggregates conjugated to nanospheres of 30, 100, and 500 nm. The Aβ internalization was quantified using Aβ‐specific 6E10 antibody. D,E) Quantification of Aβ uptake by iMGLs incubated with D) in vitro prepared aggregates of Aβ42 and Aβ40 and E) Different sized Aβ conjugated to nanospheres (30, 100, and 500 nm). (Units of uptake = integrated fluorescence intensity of the sample in the Aβ channel divided by the integrated fluorescence intensity of the corresponding Aβ42 aggregates) F) Comparison of Aβ internalization by iMGLs incubated with Aβ42 and Aβ40 conjugated to nanospheres of different sizes. (Units of uptake = integrated fluorescence intensity of the sample in the Aβ channel divided by the integrated fluorescence intensity of corresponding Aβ aggregates prepared on 30 nm nanosphere). G,H) Measurement of cytokine secretion (TNF‐α, IL‐1β, IL‐6) by iMGLs after 1 hour incubation with G) Aβ42 and H) Aβ40 conjugated nanospheres of different sizes (30, 100, and 500 nm). Cytokine release was calculated as the cytokine response to the sample divided by the cytokine response to the corresponding Aβ aggregates prepared on 30 nm nanospheres D,E) or Aβ aggregates engineered on 30 nm nanospheres F) for each replicate. I–L) Comparison of cytokine secretion by iMGLs following incubation with I) in vitro prepared Aβ42 and Aβ40 aggregates or J) 30 nm, K) 100 nm, or L) 500 nm Aβ42 and Aβ40 conjugated nanospheres. (Units of cytokine release = total cytokine measured in the media in response to the sample divided by the total cytokine measured in response to corresponding Aβ42 aggregates). Data presented as mean ± standard deviation across four biological replicates. Statistical significance was calculated via unpaired two‐sample t ‐test (D) or one‐way ANOVA with Tukey's post‐hoc test (E‐L). * P < 0.05, ** P < 0.01, *** P < 0.001, ns – non‐significant (P ≥ 0.05).

Article Snippet: To characterize the engineered protein aggregate nanospheres, we performed Bicinchoninic acid (BCA) (Figure ) and Meso Scale Discovery (MSD) (Figure , Supporting Information) assays to verify uniform protein loading across nanospheres of different sizes.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Incubation, In Vitro, Fluorescence, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Molecular Determinants of Protein Pathogenicity at the Single‐Aggregate Level

doi: 10.1002/advs.202410229

Figure Lengend Snippet: Effect of TLR4 inhibition by TAK‐242 on iMGLs' internalization of Aβ aggregates and cytokine secretion A) Schematic illustrating the protocol to assess Aβ uptake and cytokine levels using ELISA, with and without TLR‐4 inhibitor TAK‐242. B,C) Quantification of Aβ internalization by iMGLs incubated with in vitro prepared aggregates of B) Aβ42 and C) Aβ40, comparing conditions with and without TAK‐242. D,E) Comparison of Aβ uptake by iMGLs incubated with D) Aβ42 and E) Aβ40 conjugated to nanospheres of various sizes (30, 100, and 500 nm), analyzed both with and without TAK242. For each case, internalization data are normalized to conditions without TAK‐242. F,G) Measurement of cytokine secretion (TNF‐α, IL‐1β, IL‐6) by iMGLs following 1 hour incubation with in vitro prepared F) Aβ42 and G) Aβ40 aggregates, under conditions with and without TAK‐242. H–O) Measurement of cytokine release by iMGLs following 1 hour incubation with Aβ42 and Aβ40 aggregates conjugated nanospheres of different sizes: H,L) 30 nm, I,M) 100 nm, and J,N) 500 nm, with and without TAK‐242. Reduction in cytokine secretion by iMGLs incubated with K) Aβ42 and O) Aβ40 conjugated nanospheres of different sizes (30, 100, and 500 nm) following TAK‐242 treatment. For each case, cytokine release data are normalized to conditions where Aβ was added in the absence of TAK‐242. Data are presented as the mean ± standard deviation across four biological replicates. Statistical significance was assessed using an unpaired two‐sample t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns – non‐significant (P ≥ 0.05).

Article Snippet: To characterize the engineered protein aggregate nanospheres, we performed Bicinchoninic acid (BCA) (Figure ) and Meso Scale Discovery (MSD) (Figure , Supporting Information) assays to verify uniform protein loading across nanospheres of different sizes.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, In Vitro, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Molecular Determinants of Protein Pathogenicity at the Single‐Aggregate Level

doi: 10.1002/advs.202410229

Figure Lengend Snippet: Effect of Aβ40 and Aβ42 ratio on the extent of iMGLs uptake, inflammatory cytokine secretion and neuronal toxicity A) ThT assay demonstrating the aggregation kinetics of Aβ40 (30 µm), Aβ42 (3 µm) alone, and in ratios of 9:1 (Aβ40 27 µm, Aβ42 3 µm) and 7:3 (Aβ40 21 µm, Aβ42 9 µm). B) Schematic representation of SiMPull analysis for engineered protein aggregated nanosphere. C,D) Wide‐field images of fluorescent nanospheres of 30 and 100 nm, conjugated with Aβ40 and Aβ42 alone and in ratios of 9:1 and 7:3. These aggregates are captured using 10 nM biotinylated 6E10 antibody and imaged with 1 nM Aβ40 specific and Aβ42 specific antibodies. Contrast‐enhanced images are included to improve visualization where no signal from antibodies is observed. E) Quantification of colocalization and F) intensity ratio of Aβ40 coupled with Aβ42 on nanospheres at different Aβ40:Aβ42 ratios (10:0, 9:1, 7:3, 0:10). G) Representative images of iMGLs (stained with DAPI/Iba1) showing internalization of Aβ aggregates at different Aβ40:Aβ42 ratios (10:0, 9:1, 7:3, 0:10). Aβ is stained with the 6E10 antibody. H) Quantification of Aβ internalization by iMGLs incubated with in vitro prepared aggregates of different Aβ40:Aβ42 ratios. Uptake data are normalised to pure Aβ42 aggregates. I) Measurement of cytokine secretion (IL‐1β, TNF‐α, IL‐6) by iMGLs after incubation with in vitro aggregates of different Aβ40:Aβ42 ratios. Cytokine release are normalized to the secretion levels in response to pure Aβ42 aggregates. J–L) Quantification of total Aβ uptake and cytokine secretion by iMGLs following the incubation with 30 nm M,N) and 100 nm M,O) nanosphere‐conjugated Aβ aggregates of different Aβ40:Aβ42 ratios. For both uptake and cytokine release, across both nanosphere sizes, data are normalized to pure Aβ42 aggregates. M) Representative images of mouse primary neurons (stained with DAPI/acetylated Tubulin) treated with both in vitro and engineered aggregates with different Aβ40:Aβ42 ratios (10:0, 9:1, 7:3, 0:10). Aβ was detected with 6E10 antibody. N–P) Comparison of cytotoxicity, measuring LDH release from mouse primary neurons after exposure to in vitro prepared aggregates and, 30 nm O) and 100 nm P) nanospheres conjugated with different Aβ40:Aβ42 ratios. Data are normalized to values obtained when vehicle control of PBS buffers is used. Data points are plotted as the mean ± standard deviation representing three biological replicates. Statistical significance was calculated using one‐way ANOVA with post‐hoc Tukey mean comparison. * P < 0.05, ** P < 0.01, *** P < 0.001, ns – non‐significant (P ≥ 0.05).

Article Snippet: To characterize the engineered protein aggregate nanospheres, we performed Bicinchoninic acid (BCA) (Figure ) and Meso Scale Discovery (MSD) (Figure , Supporting Information) assays to verify uniform protein loading across nanospheres of different sizes.

Techniques: ThT Assay, Staining, Incubation, In Vitro, Comparison, Control, Standard Deviation

Journal: Advanced Science

Article Title: Molecular Determinants of Protein Pathogenicity at the Single‐Aggregate Level

doi: 10.1002/advs.202410229

Figure Lengend Snippet: Impact of Dutch (E22Q) and Arctic (E22G) Aβ42 missense mutations on iMGLs uptake and neuronal toxicity A,B) ThT assay illustrating the aggregation kinetics of WT Aβ42, Dutch (E22Q) Aβ42, and Arctic (E22G) Aβ42 missense mutations both alone and in 1:1 ratios with WT Aβ42. C) Schematic representation of SiMPull analysis for characterizing protein‐conjugated nanospheres. D) Wide‐field fluorescence microscopy images displaying WT Aβ42, E22Q, WT + E22Q (1:1), E22G, and WT + E22G (1:1) aggregates conjugated to 30 nm nanospheres and stained with 6E10 and 4G8 antibodies. E) Quantification of 4G8/6E10 intensity ratio for each case. F) Representative images of iMGLs (stained with DAPI/Iba1) showing internalization of different Aβ42 aggregates: WT, WT+E22Q, Aβ42 E22Q WT+E22G, and E22G. Aβ is stained with 6E10 antibody for quantification. G,H) Quantification of total Aβ uptake by iMGLs incubated with in vitro prepared aggregates and 30 nm nanosphere‐conjugated Aβ42 aggregates for WT, WT + E22Q, and WT + E22G. Uptake data are normalized to corresponding WT Aβ42 uptake. I–L) Measurement of cytokine secretion (TNF‐α in L, M and IL‐6 in N, O) by iMGLs following incubation with in vitro prepared aggregates and 30 nm nanosphere‐conjugated aggregates of WT, E22Q, E22G, WT + E22Q, and WT + E22G. Cytokine release data are normalized to responses from WT Aβ42. M) Representative images of mouse primary cortical neurons (stained with DAPI/ acetylated tubulin) treated with WT, E22Q, E22G, WT+E22Q, and WT+E22G Aβ42 aggregates. N,O) Cytotoxicity assay measuring LDH release from mouse primary cortical neurons after exposure to in vitro prepared aggregates and 30 nm nanospheres conjugated with different Aβ aggregates. Data are normalized to values from PBS buffer used as the vehicle control. Data presented as mean ± standard deviation across four biological replicates. Statistical significance was assessed via unpaired two‐sample t ‐test or one‐way ANOVA with Tukey's post‐hoc test * P < 0.05, ** P < 0.01, *** P < 0.001, ns non‐significant (P ≥ 0.05).

Article Snippet: To characterize the engineered protein aggregate nanospheres, we performed Bicinchoninic acid (BCA) (Figure ) and Meso Scale Discovery (MSD) (Figure , Supporting Information) assays to verify uniform protein loading across nanospheres of different sizes.

Techniques: ThT Assay, Fluorescence, Microscopy, Staining, Incubation, In Vitro, Cytotoxicity Assay, Control, Standard Deviation

Journal: Advanced Science

Article Title: Molecular Determinants of Protein Pathogenicity at the Single‐Aggregate Level

doi: 10.1002/advs.202410229

Figure Lengend Snippet: The proportion of phosphorylated αSyn within αSyn aggregates impacts its ability to permeabilize mitochondria mimicking membrane. A) Schematic representation of the SiMPull analysis used for characterizing αSyn and pSyn‐conjugated nanospheres. B) Three‐color epi‐fluorescence images depicting various ratios of αSyn and pSyn (100:0, 75:25, 50:50, 25:75, and 0:100) engineered on 30 nm fluorescent nanospheres (1 µM monomer equivalents) using 10 nM biotinylated Syn211 antibody for capture and αSyn confirmation specific Alexa‐561‐fluor conjugated 5 nM MJFR‐14‐6‐4‐2 antibody in conjunction with pSyn‐specific 5 nM Alexa‐637‐fluor labelled Anti‐Alpha‐synuclein (phospho S129) antibody EP1536Y. C) Quantification of the three color colocalization of nanosphere, αSyn and pSyn at various αSyn and pSyn ratios. D) The intensity ratios between pSyn and αSyn channels gradually increase as the proportion of pSyn within the αSyn aggregates rises. The intensity ratio of pSyn to αSyn is calculated by comparing the corresponding antibody intensities. E) Schematic illustration of the experimental protocol for quantifying membrane permeabilization by measuring Ca 2+ ‐ion influx in response to aggregates. F) Representative images show the Ca 2+ ‐ion influx into Cal‐520 dye‐filled that are sequentially treated with buffer, sample, and ionomycin. (G‐I) Quantification of Ca 2+ ‐ion influx in individual vesicles, composed with and without cardiolipin, in response to aggregates composed of various αSyn and pSyn ratios. G) Comparison of in vitro prepared aggregates using pure αSyn and pSyn. H,I) Quantification of the Ca 2+ ‐ion influx into thousands of individual vesicles composed of 20% Cardiolipin I) and without Cardiolipin H), responding to varying ratios of αSyn to pSyn (0%, 25%, 50%, 75%, and 100%) engineered on the surface of 30 nm nanospheres. All Ca 2+ ‐ion influx measurements are normalized to the response from WT αSyn aggregates. Data points represent the mean ± standard deviation from three independent replicates. Statistical significance was assessed using two‐samples t ‐test (L) or one‐way ANOVA followed by Tukey's post‐hoc mean comparison. * P < 0.05, ** P < 0.01, *** P < 0.001, ns – non‐significant ( P ≥ 0.05).

Article Snippet: To characterize the engineered protein aggregate nanospheres, we performed Bicinchoninic acid (BCA) (Figure ) and Meso Scale Discovery (MSD) (Figure , Supporting Information) assays to verify uniform protein loading across nanospheres of different sizes.

Techniques: Membrane, Fluorescence, Comparison, In Vitro, Standard Deviation