biotinylated anti human egfr  (R&D Systems)

Journal: Advanced Science

Article Title: Distinct CTC Specific RNA Profile Enables NSCLC Early Detection and Dynamic Monitoring of Advanced NSCLC

doi: 10.1002/advs.202417849

Figure Lengend Snippet: NSCLC characteristic mRNA selection and optimization of CTC purification. A) Illustration of NSCLC specific mRNA selection. B) Expression levels of 6 genes in PBMCs and four NSCLC cell lines. C) Expression levels of 6 genes in PBMCs isolated from different patients. D) Illustration of CTC purification using artificial samples. Optimization of concentrations of E) anti‐EpCAM, F) anti‐EGFR, and G) anti‐N‐cadherin for CTC capture. H) A respective SEM image of an isolated cancer cell with mounts of MNPs on its surface. I) The relative expression levels of EpCAM, EGFR, and N‐cadherin on these four cell lines were analyzed by flow cytometry. J) Capture performance of the antibody cocktail‐modified MNPs for the four cancer cell lines.

Article Snippet: Multi‐antibody‐modified MNPs were prepared as follows: 0.1 mg of streptavidin‐modified beads (Beaver) were incubated for 30 min with a mixture of antibodies, including biotinylated anti‐human EpCAM (R&D Systems, polyclonal goat IgG, 200 ng/200 μL), biotinylated anti‐human EGFR (R&D Systems, polyclonal goat IgG, 200 ng/200 μL), and biotinylated anti‐human N‐cadherin (Proteintech, polyclonal rabbit IgG, 200 ng/200 μL).

Techniques: Selection, Purification, Expressing, Isolation, Flow Cytometry, Modification

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Schematic representation of anti-EGFR CAR lentiviral LentiONE expression vector (GEGTech). The CAR construct contains an anti-EGFR scFv fused to a CD8a hinge and transmembrane domain, a 4-1BB intracellular, and a CD3ζ signaling domain. SP—signaling peptide leader sequence. The CAR is expressed from a PGK promoter, while the GFP (green fluorescent protein) reporter gene is expressed from a miniCMV promoter, part of the same bidirectional promoter unit. 5′,3′-LTR—long tandem repeats; Ψ—RNA packaging signal; cPPT—central polypurine tract; polyA—polyadenylation signal; WPRE—woodchuck hepatitis promoter regulatory element.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing, Plasmid Preparation, Construct, Sequencing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: ( A ) Transduction efficiency assessed by the flow cytometry analysis of GFP-expressing cells at 3 days post-transduction. In the TNK-polarized group (stimulated with cytokines and beads), a higher transduction efficiency was observed (83 ± 8% vs. 26 ± 9%, p = 0.001). ( B , C ) Dot plot graphs of CAR-NK-polarized cells ( B ) and CAR-TNK-polarized cells ( C ); cells were treated with biotinylated EGFR and streptavidin-APC. The upper right quadrant (UR) indicates the population of CAR+ cells (FITC-positive and APC-positive); **— p < 0.01.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Transduction, Flow Cytometry, Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: ( A ) Dot-plot analysis of MICA/B expression in MDA-MB468 (red) cells vs. SK-BR3 (blue) cells showed a similar expression profile. ( B ) The MDA-MB468 cell line (red) exhibited a higher EGFR expression compared to SK-BR3 cells (blue). ( C ) CD1d expression in both cell types is reduced, but with a slight increase in the MDA-MB468 cell line. ( D ) CD95 (Fas) expression in MDA-MB468 cells (red) vs. SK-BR3 (blue) exhibited a similar expression profile in both cell types.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: Short-term cytotoxic activity of anti-EGFR CAR transduced cells compared to untransduced cells against calcein-labeled target MDA-MB-468 cells ( A ), SK-BR3 cells ( B ), K562 cells ( C ), and allogeneic PBMCs ( D ). The transduction significantly increased the effector cells’ cytotoxic activity, which correlated with EGFR expression levels in EGFR-expressing tumor cell lines MDA-MB-468 (E:T = 10:1—34 ± 4% vs. 5 ± 1%, p < 0.001; E:T = 5:1—25 ± 2% vs. 3 ± 1%, p < 0.05; E:T = 1:1—10 ± 0.5% vs. 1 ± 0.5%, p < 0.05) and SK-BR-3 (E:T = 10:1—24 ± 2% vs. 6 ± 1%, p < 0.05; E:T = 5:1—17 ± 0.2% vs. 5 ± 0.2%, p < 0.001; E:T = 1:1—3 ± 1% vs. 2 ± 1%, p = 0.28), but without statistical significance in EGFR-negative, NK-sensitive K562 cells, nor allogeneic PBMCs (E:T = 1:1—3.58 ± 2.53% vs. 2.74 ± 1.90%, p = 0.66; E:T = 5:1—0.79 ± 0.26% vs. 0.84 ± 0.62%, p = 0.90; E:T = 10:1—0.99 ± 0.79% vs. 1.06 ± 0.53%, p = 0.90); ns—not significant ( p > 0.05); **— p < 0.01; *** p < 0.001.

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Activity Assay, Labeling, Transduction, Expressing

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: ( A ) RTCA analysis of EGFR-expressing MDA-MB468 cells incubated for 24 h with transduced anti-EGFR CAR cells (red) and untransduced cells (blue). ( B ) RTCA analysis of the EGFR positive SK-BR3 cell line, incubated for 24 hours with CAR-expressing cells (red) and untransduced PBMCs (blue). In the first 2 h, a rapid decline in cell index was observed in both cell lines treated with CAR cells (red). However, this effect was more pronounced in the MDA-MB-468 group. A poor decline was also observed in the untransduced group (blue), which may account for non-specific lysis. The control group experienced constant proliferation (black).

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Expressing, Incubation, Lysis, Control

Journal: Biomedicines

Article Title: Exploring CAR-PBMCs: A Novel Strategy Against EGFR-Positive Tumor Cells

doi: 10.3390/biomedicines13020264

Figure Lengend Snippet: ( A ) Cell index slopes for MDA-MB468 cells incubated with CAR-expressing and untransduced cells (UnTD). ( B ) Cell index slopes for SK-BR3 cells treated with CAR-expressing cells and untransduced (UnTD) cells. The slopes of the cell indices (CI slope) were more negative in the CAR-treated target cells compared to untransduced cells. In the MDA-MB-468 control cells ( A ), the slope had an overall positive value, suggestive for cell proliferation, compared to a negative value in the SK-BR3 control group, which may reflect the differences in EGFR expression as a mechanism for cell survival, as well as an increased expression of the non-specific activator molecule MICA/B in SK-BR3. The CI slopes for tumor cells incubated with CAR-PBMCs were comparable, with net negative values (MDA-MB468: −0.076 vs. SK-BR3: −0.074, p = 0.1714).

Article Snippet: CAR expression assessment was performed by labeling the transduced cells with biotinylated EGFR (R&D Systems, Minneapolis, MN, USA, cat. #BAF231), followed by APC-conjugated streptavidin (BioLegend, San Diego, CA, USA, cat. #405207), according to the manufacturer’s protocol, and subsequent flow cytometric analysis.

Techniques: Incubation, Expressing, Control

Journal: ACS sensors

Article Title: Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis.

doi: 10.1021/acssensors.2c01750

Figure Lengend Snippet: Figure 2. EVPio assay optimization for inner protein detection. (A) Illustration of the need for an AR step. (i) The inner proteins of captured EVs are bound by the lipid bilayer membrane and inaccessible to antibodies. (ii) Fixation cross-links inner proteins, while permeabilization allows antibodies to diffuse into EVs, but methylene cross-linking limits the accessibility to epitopes. Permeabilization can be used to allow antibodies to breach the membrane while keeping cytosolic proteins in place, but cross-links can limit the epitope accessibility. (iii) Heat-based AR treatment of EVs induces partial hydrolytic breakage of cross-links, allowing antibody binding of retained and immobilized proteins. (B) AR optimization experiments with different buffers/additives, temperatures and incubation times, and fluorescence detection signal for inner protein HSP90 and outer protein EGFR, with superposed heatmap. The red rectangle indicates the optimal condition that was chosen based on high HSP90 and high EGFR signals. Signal values are above the threshold of NC + 2SD unless grayed. A zero value indicates a negative or zero difference between the signal and its associated no EV control. (C) Fluorescence micrograph of the optimal AR condition: 1 min at 90 °C, in PBS. (D) Bar graph of HSP90 detection signal on spots with CD63 capture antibody and negative control spots with GAM antibody serving as NC. The threshold of NC + 2SD is illustrated as a horizontal line.

Article Snippet: 2022, 7, 3817−3828 3825 Invitrogen), and biotinylated EGFR antibodies (BAF231, R&D Systems) were used at the detection step at concentrations of 5, 5, and 1 μg/mL, respectively, followed by labeling with Alexa Fluor 647- conjugated goat anti-rabbit antibodies (A-21245, Invitrogen) and streptavidin (S21374, Invitrogen).

Techniques: Membrane, Binding Assay, Incubation, Fluorescence, Control, Negative Control