Journal: Frontiers in cell and developmental biology

Article Title: Effect of AMH on primordial follicle populations in mouse ovaries and human pre-pubertal ovarian xenografts during doxorubicin treatment.

doi: 10.3389/fcell.2024.1449156

Figure Lengend Snippet: FIGURE 2 Treatment regimen for administration of anti-Müllerian hormone (AMH) and/or doxorubicin (DXR) to SCID mice (severe combined immunodeficient) containing human ovarian transplants. Ten days after human ovarian tissue was xenotransplanted into SCID mice, animals were intraperitoneally (IP) injected daily with PBS or rhAMH for 8 days with an additional single IP injection of PBS or DXR on the second day.

Article Snippet: Recombinant human AMH (rhAMH) was purchased from R&D Systems (1737-MS-010/CF, United Kingdom).

Techniques: Injection

Journal: Frontiers in cell and developmental biology

Article Title: Effect of AMH on primordial follicle populations in mouse ovaries and human pre-pubertal ovarian xenografts during doxorubicin treatment.

doi: 10.3389/fcell.2024.1449156

Figure Lengend Snippet: FIGURE 3 Analysis of mouse ovarian follicles in cultured pre-pubertal ovaries treated with DXR and AMH stained with H and E. (A) Representative images of morphologically healthy follicles (scale bar = 25 μm): i: Primordial follicles. ii-iv: Growing follicles (ii: Transitional; iii: Primary; iv: Secondary). (B) Unhealthy follicles (scale bar = 100 μm): black arrows denote pyknotic granulosa cells, red arrows denote atretic oocytes. (C) Total number of morphologically healthy primordial follicles in mouse ovaries treated with different concentrations of DXR. Values are presented as Mean ± S.E.M. Control n = 3; 50 ng/mL DXR n = 3; 100 ng/mL DXR n = 5; 200 ng/mL DXR n = 3. (D) Number of morphologically healthy primordial follicles in mouse ovaries cultured with different concentrations of rhAMH and with or without exposure to DXR. Values represent Mean ± S.E.M ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Article Snippet: Recombinant human AMH (rhAMH) was purchased from R&D Systems (1737-MS-010/CF, United Kingdom).

Techniques: Cell Culture, Staining, Control

Journal: Journal of neurochemistry

Article Title: Macrophage stimulating protein preserves blood brain barrier integrity after intracerebral hemorrhage through recepteur d'origine nantais dependent GAB1/Src/β-catenin pathway activation in a mouse model.

doi: 10.1111/jnc.14622

Figure Lengend Snippet: Fig. 1 Time-line of study design. Representative figure showing experimental design and number of animals for each group. ICH, intracerebral hemorrhage; WB, western blot; BBB, blood brain barrier; rhMSP, recombinant human macrophage stimulating protein.

Article Snippet: Eight-week male CD1 mice were pseudo randomly assigned to the following groups: Sham, ICH + Vehicle, ICH + rhMSP (MSP-0.1 lg), ICH + rhMSP (MSP-0.3 lg), ICH + rhMSP (MSP-1 lg), ICH + rhMSP + scrambled siRNA, ICH + rhMSP + RON siRNA, ICH + rhMSP + GAB1 siRNA. rhMSP (6244-MS-025/CF; R&D Systems, Minneapolis, MN, USA) was administered via intranasal route 1 h post-ICH and then once daily for 3 days to the 72 h groups.

Techniques: Western Blot, Recombinant

Journal: Nature Communications

Article Title: Chimeric antigen receptor macrophages (CAR-M) sensitize HER2+ solid tumors to PD1 blockade in pre-clinical models

doi: 10.1038/s41467-024-55770-1

Figure Lengend Snippet: a PCA clustering from HSCs, monocytes, and CAR-M cultures ( n = 3). b Incucyte-based killing assays of UTD-M or CAR-M generated from monocytes (GM-CSF or M-CSF) and HSCs (M-CSF) cocultured with AU565 NLG at effector:target (E:T) ratio 1:1 for 40 h. Green fluorescence intensity (GFI) values were normalized to the 0 h timepoint, then further normalized to the target cells. Values were transformed to % cytotoxicity (1-GFI)*100 %). Data are representative of three experiments and shows the mean ± SD of n = 4 technical replicates. Statistical analysis was calculated using the ordinary one-way ANOVA test. c Viability, and sustained Ad5f35 CAR expression up to 13 days post-transduction of M-CSF-derived, HSCs-differentiated macrophage. Data are representative of several experiments and show the mean ± SEM of n = 3 technical replicates. d , Representative FACS plots showing CAR expression. e , f Expression of pro-inflammatory (e.g., CD80, CD86 and MHC-II) and anti-inflammatory (e.g., CD206) markers were measured 48 h. after macrophages transduction at the protein level ( e , n = 3 technical replicates) by flow cytometry and at the gene level ( f , n = 3 biological replicate) by RNA-Seq. Statistical analysis for ( e ) was calculated with the two-sided paired t test. g Killing assays with UTD-M or CAR-M cocultured with target (MC38-HER2 and CT26-HER2) and parental cell lines (WT, HER2 negative) at E:T 2:1. GFI values were first normalized to the 0 h. timepoint then normalized to the respective target cells. Data is shown as mean ± SEM of n = 2–3 technical replicates. Statistical significance was calculated via two-way RM ANOVA. Graph is representative of three experiments. h , i Cytokine and chemokine production by CAR-M after overnight stimulation with plate-bound recombinant human HER2 protein or recombinant mesothelin (Meso; irrelevant antigen control) was determined by cytokine multiplex assay ( n = 3 biological replicate). h Data are represented as the mean ± SD. Statistical analysis was performed using the two-way ANOVA Tukey’s multiple comparisons test. i Heatmap comparing mesothelin- vs HER2-stimulated CAR-M cytokine and chemokine production. Two-sided unpaired Student’s t test was used to calculate significance. j Phagocytosis assay of MC38 cells by UTD-M or CAR-M determined by flow cytometry. Data represents the mean ± SEM of n = 3 technical replicates. Statistical significance was calculated via two-way ANOVA multiple comparisons. Graph is representative of two experiments. k MHC class I expression on MC38WT and MC38-HER2 cells co-cultured with UTD-M or CAR-M. Data show the mean ± SEM of n = 2 technical replicates and representative FACS plots show MHC-I (H2kB) MFI. Graph is representative of two experiments. l Killing kinetics of UTD-M and CAR-M co-cultured with MC38OVA + HER2 + and transgenic CD8 + T cells (OT−1), at E:T:T (Effector:Target:T cells), 2:1:6. GFI values were normalized to the 0 h. timepoint and then, further normalized to the target cells. Data represents the mean ± SEM of n = 3 technical replicates and is representative of two experiments. m UTD-M and CAR-M were co-cultured with MC38OVA+ and transgenic T cells (OT-1), at E:T:T (Effector:Target:T cells), 2:1:6. Flow cytometry was used to assess T cell activation at the 24 h timepoint ( n = 3 technical replicates). Fold change was calculated by dividing the % expression of CAR-M by that of UTD-M. For all images: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human Her2 (target antigen; 10004-H08H, SinoBiological) or human mesothelin (irrelevant antigen control; 3265-MS, R&D Systems) were resuspended in PBS to a concentration of 28 nM.

Techniques: Generated, Fluorescence, Transformation Assay, Expressing, Transduction, Derivative Assay, Flow Cytometry, RNA Sequencing, Recombinant, Control, Multiplex Assay, Phagocytosis Assay, Cell Culture, Transgenic Assay, Activation Assay

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of HFt with AcLDL in human macrophages. hMDM were incubated simultaneously with HFt-AF488 (50 μg/ml) (green) and AcLDL-AF594 (5 μg/ml) (red) for 15 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and AcLDL (yellow foci) for hMDM. Scale bar = 20 μm (10 µm in zoomed region). b Flow cytometry analysis of internalized HFt-AF488 (5 μg/ml) by hMDM in the absence or presence of AcLDL at indicated concentrations within 30 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 3 different donors. c Representative confocal microscopy images of internalized HFt-AF488 (5 μg/ml) (green) by hMDM in the absence or presence of AcLDL (100 μg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. d Flow cytometry analysis of internalized AcLDL-AF488 (1 μg/ml) by hMDM in the absence or presence of HFt at indicated concentrations within 30 min at 37 °C. Data are presented as mean fluorescence intensity (MFI) of AcLDL-AF488. Data are presented as mean ± SEM from n = 3 different donors. e Representative confocal microscopy images of internalized AcLDL-AF488 (1 μg/ml) (green) by hMDM in the absence or presence of HFt (2,5 mg/ml) within 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. f–h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I), poly(G) and poly(C) (control) ( f ), fucoidan and mannan (control) ( g ) and dextran sulfate or chondroitin sulfate (control) ( h ) Flow cytometry data are presented as % of HFt-AF488 uptake in untreated, control cells. Data are presented as mean ± SEM from n = 3 ( f , h ) or 6 ( g ) different donors (hMDM). i–k Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with indicated concentrations of various ligands of class A scavenger receptors or structurally related ligands that do not bind to this group of receptors (negative controls): poly(I) and poly(C) (control) ( i ), fucoidan and mannan (control) ( j ) and dextran sulfate or chondroitin sulfate (control) ( k ). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. l Quantitative real-time PCR analysis of MSR1, SCARA5, and MARCO mRNA expression in hMDM. Data are presented as mean ± SEM from n = 3 different donors (hMDM). m Western blot analysis of MSR1 protein expression in monocytes and hMDM. Representative western blot images are shown. n Flow cytometry analysis of MSR1 cell surface staining in hMDM upon stimulation with HFt (200 µg/ml) for indicated time points at 37 °C. For comparison, untreated cells were used (Control). Data are presented as mean ± SEM % of MSR1 expression in control cells, n = 3 independent donors (hMDM). o Western blot analysis of MSR1 protein expression in hMDM that were untreated (Control) or treated with cycloheximide (CHX) (20 µg/ml) for 1 h prior to HFt stimulation (200 µg/ml) (CHX+HFt) for the indicated time points at 37 °C. For comparison, cells treated only with CHX were used. Representative western blot images are shown. p Quantitative analysis of relative MSR1 expression in hMDM shown in ( o ). Data are presented as mean ± SEM from n = 3 independent replicates. q Western blot analysis showing MSR1 expression in either untransfected hMDM (Control) or cells transfected with one of the following siRNA sequences: scramble siRNA (siScr), no. 1 siRNA targeting MSR1 (siMSR1-1), no. 2 siRNA targeting MSR1 (siMSR1-2) at 72 h after transfection. r Quantitative analysis of western blot for MSR1 expression shown in ( q ). Data are presented as mean ± SEM % of MSR1 expression in control cells (Control), n = 3 different donors (hMDM). s and ( t ) Flow cytometry analysis of internalized AcLDL-AF488 (5 µg/ml) ( s ) or HFt-AF488 (100 μg/ml) ( t ) by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as mean ± SEM % of ligand uptake in control cells (Control), n = 3 different donors. u and ( v ) Representative confocal microscopy images of internalized AcLDL-AF488 (5 μg/ml) (green) ( u ) and HFt-AF488 (50 μg/ml) (green) ( v ) within 30 min at 37 °C by THP-1 macrophages after MSR1 gene-knockdown. Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. w Western blot analysis of MSR1 protein expression in hMDM macrophages polarized to M1 or M2 phenotypes. Representative western blot images are shown. x Relative expression of MSR1 in M1 and M2 hMDM macrophages quantified based on western blot analysis shown in ( w ). Data are presented as mean ± SEM from n = 4 different donors. y Flow cytometry analysis of internalized HFt-AF488 by M1 and M2 hMDM macrophages given at 25 µg/ml within 20 or 60 min at 37 °C. Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 different donors. The one-way ANOVA and Dunnett’s post-hoc test were used for statistical analysis in panels b , d , r – t . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panels f – h . The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis in panels ( p , y ). The Student’s t -test was used for statistical analysis in panel ( x ). For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Comparison, Transfection, Knockdown

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Representative western blot images are shown. b and ( c ) Flow cytometry analysis of cell surface expression of MSR1 in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2) or the plasmid encoding the MSR1 gene (p-MSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of BV421. Data are presented as mean ± SEM from n = 3 independent replicates. d Flow cytometry analysis of internalized AcLDL-AF488 (5 μg/ml) and HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2. Flow cytometry data are presented as mean fluorescence intensity (MFI) of fluorescently labeled ligands. Data are presented as mean ± SEM from n = 3 independent replicates. e , f Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) ( e ) and AcLDL-AF488 (5 μg/ml) ( f ) (green) within 30 min at 37 °C by HEK293 cells, transfected with either p-mCherry2 or p-MSR1-mCherry2 (red). Afterwards, cells were fixed and stained with Hoechst 33342 (blue). Scale bar = 20 μm. g Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.022–50 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. h Competitive effect of unlabeled HFt on AlphaScreen signal generated by His-MSR1 and Biotin-HFt interaction. Fixed concentrations of 4 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( g ) in the presence of increasing concentration of unlabeled HFt (0.076–1500 nM, 3-fold serial dilution). IC50 potency of unlabeled HFt in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. i Spectral shift dose–response curve between unlabeled HFt and His-MSR1 conjugated with the RED-tris-NTA dye. Data was analyzed using software provided by the manufacturer and is presented as the emission fluorescence ratio 670/650 vs. log(ligand concentration). K d value was calculated by fitting data with a 1:1 binding model. Data presented as mean ± SEM from n = 5 independent replicates. j Schematic representation of the full-length MSR1 and SRCR domain deletion mutant of MSR1. Created in BioRender. Taciak, B. (2024) https://BioRender.com/a34q680 . k Western blot analysis of MSR1 protein expression in HEK293 cells at 24 h after transfection with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Representative western blot images are shown. l and ( m ) Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene (p-ΔMSR1-mCherry2). Flow cytometry data are presented as the mean fluorescence intensity (MFI) of HFt-AF488. Data are presented as mean ± SEM from n = 4 independent replicates. n Representative confocal microscopy images of internalized HFt-AF488 (50 μg/ml) within 30 min at 37 °C by HEK293 cells, transfected with the control plasmid (p-mCherry2), the plasmid encoding the full-length MSR1 gene (p-MSR1-mCherry2) or the plasmid encoding SRCR domain deletion mutant of MSR1 gene ( p -ΔMSR1-mCherry2) (red). Nuclei were stained with Hoechst 33342 (blue). Scale bar = 20 μm. The two-way ANOVA followed by Sidak’s multiple comparisons post hoc test was used for statistical analysis. For all panels, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Flow Cytometry, Fluorescence, Labeling, Confocal Microscopy, Staining, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, Software, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Harnessing macrophage-drug conjugates for allogeneic cell-based therapy of solid tumors via the TRAIN mechanism

doi: 10.1038/s41467-025-56637-9

Figure Lengend Snippet: a Representative confocal microscopy images of colocalization of the 735 drug with HFt-735 in human macrophages. The hMDM were incubated with HFt-AF488 (50 μg/ml) or HFt-735-AF488 for 15 min at 37 °C, fixed, and stained with the anti-735 antibody and Hoechst 33342 (blue). Merged fluorescence images show colocalization of HFt and 735 drug (yellow foci). Scale bar = 20 μm. b and ( c ) Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (5 μg/ml) by hMDM ( b ) or THP-1 macrophages ( c ) in the absence or presence of AcLDL within 30 min at 37 °C. Flow cytometry data are presented as mean fluorescence intensity (MFI) of HFt-AF488 or HFt-735-AF488. Data presented as mean ± SEM from n = 3 different donors ( b , hMDM) or n = 3 independent replicates ( c , THP-1). d Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 different donors (hMDM). e Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by hMDM untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. f Flow cytometry analysis of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C with poly(G) binding to scavenger receptor and poly(C) (control). Flow cytometry data are presented as % of HFt-AF488 and HFt-735-AF488 uptake in untreated, control cells. Data presented as mean ± SEM from n = 3 (HFt-AF488) and n = 3 (HFt-735-AF488) independent replicates. g Representative confocal microscopy images of internalized HFt-AF488 and HFt-735-AF488 (50 μg/ml) (green) within 30 min at 37 °C by THP-1 cells untreated or pre-treated for 30 min at 37 °C ligand of class A scavenger receptor or structurally related ligand that does not bind to this group of receptors (negative control): poly(G) and poly(C) (control). Afterwards cells were fixed and stained with Hoechst 33342 (blue) and anti-735 drug antibody. Scale bar = 20 μm. h Flow cytometry analysis of internalized HFt-AF488 (50 μg/ml) and HFt-735-AF488 by hMDM after MSR1 gene-knockdown within 30 min at 37 °C. For comparison, untreated cells (Control) and cells treated with a negative, scramble control siRNA (siScr) were used. Flow cytometry data are presented as % of ligand uptake in control cells (Control). Data presented as mean ± SEM from n = 3 different donors (hMDM). i Optimization of AlphaScreen assay conditions by cross-titration of His-MSR1 (0.41–100 nM, 3-fold serial dilution) and Biotin-HFt (0.008–18 nM, 3-fold serial dilution) at fixed 10 μg/ml concentration of both nickel chelated acceptor and streptavidin donor beads. Data shown are from a single pilot run conducted for assay optimization. j Competitive effect of HFt-735 on AlphaScreen signal generated by His-MSR1 & Biotin-HFt interaction. Fixed concentrations of 8 nM His-MSR1 and 2 nM Biotin-HFt were used below the hook point of the bead assay ( i ) in the presence of increasing concentration of HFt-735 (0.006–1000 nM, 3-fold serial dilution). IC 50 potency of HFt-735 in displacing Biotin-HFt from interaction with His-MSR1 was calculated by fitting it to a four-parameter nonlinear regression. Data presented as mean ± SEM from n = 3 independent replicates. k In vitro evaluation of cancer cell killing by MDC-735 against MDA-MB-231, BxPC-3, SK-OV-3 and A549 cancer cells in co-culture. Macrophages were incubated with 10, 100, 1000 μmol of HFt-735 complex or plain medium (0). Data are presented as mean ± SEM of n = 3 independent replicates. l Representative confocal microscopy images captured after 24 h co-culture of MDC-735 (pre-labeled with CellTrace Violet CT) with SK-OV-3 cancer cells (pre-labeled with CellTrace Far Red). Cells fixed after 24 h were additionally stained with anti-735 (green) and anti-HFt (red) antibodies. Co-localization of green and red signals both in macrophages and in cancer cells are pointed out by the arrowheads. Scale bar = 10 µM. The one-way ANOVA with Dunnett’s post-hoc test was used for statistical analysis in panels b – d , f , h . The two-way ANOVA and Tukey’s post-hoc tests were used for statistical analysis in panel k . For all panels, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: Biotin-HFt, His-tagged MSR1 (His-MSR1, R&D Systems, 2708-MS-050), nickel chelated acceptor and streptavidin-coated donor beads (PerkinElmer, 6760619C) were diluted in assay buffer consisting of PBS (Biowest, L06151000), 1 mg/ml BSA (Sigma-Aldrich, A3294) and 0.05% Tween 20 (Sigma-Aldrich, P1379).

Techniques: Confocal Microscopy, Incubation, Staining, Fluorescence, Flow Cytometry, Binding Assay, Control, Negative Control, Knockdown, Comparison, Amplified Luminescent Proximity Homogenous Assay, Titration, Serial Dilution, Concentration Assay, Generated, In Vitro, Co-Culture Assay, Labeling

Journal: JCI insight

Article Title: Affinity-tuned mesothelin CAR T cells demonstrate enhanced targeting specificity and reduced off-tumor toxicity.

doi: 10.1172/jci.insight.186268

Figure Lengend Snippet: Figure 2. Binding affinities of MSLN CAR variants. (A) Schematic representation of the lentiviral vectors containing MSLN CAR variants. (B) Relative binding of MSLN CAR variants was estimated by staining CAR-expressing Jurkat T cells with 25 nM of Alexa Fluor 647–conjugated monomeric human and mouse MSLN, using NT cells as control. (C) Determination of the equilibrium binding affinities of CAR variants using a saturation binding assay with Alexa Fluor 647–conjugated human and mouse MSLN (n = 1).

Article Snippet: Two alpacas (obtained from local alpaca farms by Tufts University) were immunized with 5 subcutaneous injections of the C-terminal domains of both recombinant human MSLN (aa 296–580, R&D Systems, Bio-Techne, catalog 3265-MS) and mouse MSLN (aa 298–600, R&D Systems, Bio-Techne, catalog 8604-MS).

Techniques: Binding Assay, Staining, Expressing, Control, Saturation Assay