biotin conjugated goat polyclonal anti mouse igg  (Danaher Inc)

Journal: eLife

Article Title: Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

doi: 10.7554/eLife.78330

Figure Lengend Snippet: ( A ) Transmission image of a chamber of the microfluidic chip containing the traps. Scale bar 100 µm. Inset: cell–droplet doublet in a microfluidic trap. Bright-field image and fluorescence image (nucleus: cyan; antigen: gray). Scale bar 5 µm. ( B ) Schematic representation of the surface of an oil droplet used for antigen presentation. ( C ) Time-lapse images of antigen recruitment on a F(ab′) 2 αIgG-coated droplet (acting as an antigen). Scale bar 5 µm. ( D ) Schematic representation of the quantification of antigen recruitment at the immune synapse. ( E ) Quantification over time of recruitment on BSA-coated (negative control) or αIgG-coated droplets at the immune synapse (median ± IQR) and ( F ) plateau of antigen recruitment (average value 25–30 min) on BSA- or αIgG-coated droplets (mean ± SEM, BSA N = 14;7, αIgG N = 4;15;4;4, pooled from >2 independent experiments, Mann–Whitney test). Figure 1—source data 1. Data tables related to graphs in .

Article Snippet: Antibody , Biotin-SP-conjugated F(ab′) 2 goat polyclonal anti-mouse IgG , Jackson ImmunoResearch , 115-066-072 , Droplet functionalization (5.7 µL).

Techniques: Transmission Assay, Fluorescence, Negative Control, MANN-WHITNEY

Journal: eLife

Article Title: Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

doi: 10.7554/eLife.78330

Figure Lengend Snippet: Experiments for this figure were performed using IIA1.6 cells, stained with SiRTubulin and Hoechst to visualize the centrosome and the nucleus, in contact with a F(ab′) 2 αIgG-coated droplet, imaged with SDCM 3D and quantified in 3D. Cells were pretreated for 1 hr either with DMSO or with latrunculin A 2 µM, kept in solution during the experiment. ( A ) Plateau of antigen recruitment (average values 25–30 min). Line at antigen recruitment = 1 (uniform fluorescence on the droplet). Median ± IQR, DMSO N = 7;10, LatA N = 6;18, two independent experiments, Mann–Whitney test (quantification: see ). ( B ) Maximum diacylglycerol (DAG) enrichment (in 0–10 min). Median ± IQR, DMSO N = 1;5;4, LatA N = 2;5;2, three independent experiments, Mann–Whitney test (quantification: see ). ( C ) Time-lapse images of untreated (DMSO) or LatA-treated cells, centrosome in red, nucleus in blue, and antigen in gray. Scale bar 5 µm. Right: angle between the cell–droplet axis and the cell–nucleus invagination (blue) or cell–centrosome (red) axis in time (quantification: see ). ( D ) Nucleus–droplet distance over time. Mean ± SEM, DMSO N = 7;10, LatA N = 15;17, two independent experiments. ( E ) Average centrosome–droplet distance (25–30 min). Median ± IQR, DMSO N = 6;10, LatA N = 11;17, two independent experiments, Mann–Whitney test. ( F ) Time of centrosome polarization (threshold distance <2 µm). Median ± IQR, DMSO N = 4;6, LatA N = 4;5, two independent experiments, Mann–Whitney test. ( G ) Nucleus orientation and centrosome orientation (quantification: see ) during the first 15 min, for DMSO-treated cells. N = 6;10 cells, one image every 30 s, two independent experiments. Nonparametric Spearman correlation between nucleus–centrosome pairs of data, average correlation 0.93, confidence interval: 0.86–0.97. Figure 4—source data 1. Data tables related to graphs in .

Article Snippet: Antibody , Biotin-SP-conjugated F(ab′) 2 goat polyclonal anti-mouse IgG , Jackson ImmunoResearch , 115-066-072 , Droplet functionalization (5.7 µL).

Techniques: Staining, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

doi: 10.7554/eLife.78330

Figure Lengend Snippet: Experiments for this figure were performed using IIA1.6 cells transfected either with siCtrl or siGEF-H1 siRNAs 60 hr before experiment, with F-tractin-tdTomato the day before experiment, then put in contact with a F(ab′) 2 αIgG-coated droplet. Cells were pretreated for 1 hr with DMSO, suberoylanilide hydroxamic acid (SAHA) 10 µM or with nocodazole 5 µM, kept in solution during the experiment. ( A ) Quantification of the ratio of acetylated α-tubulin/α-tubulin in the whole cell, for IIA1.6 cells in contact with a droplet for different times, by immunofluorescence. Imaging by confocal microscopy (median ± IQR, 0–5 min N = 14;20, 15–20 min N = 18;14, two independent experiments, Mann–Whitney test). ( B ) Left: immunofluorescence images of IIA1.6 cells treated with DMSO or nocodazole, and in contact with a droplet for 0–5 min or 15–20 min. F-actin stained with phalloidin (red), GEF-H1 (green), and antigen on droplet (blue). Scale bar 6 µm. Right: quantification of the enrichment in GEF-H1 within 1 µm of the droplet divided by the total intensity in the cell in one plane, imaged by laser scanning confocal microscopy (LSCM), for IIA1.6 cells in contact with a droplet for different times, by immunofluorescence (median ± IQR, DMSO 0–5 min N = 20;18, DMSO 15–20 min N = 20;20, Noco 15–20 min N = 19;20, two independent experiments, Kruskal–Wallis test with multiple comparisons, Dunn’s post test). ( C ) From immunofluorescence imaged with LSCM, quantification of the enrichment in GEF-H1 within 1 µm of the droplet divided by the total intensity in the cell, in one plane, and ( D ) quantification of F-actin (stained with phalloidin) on six planes (δz = 0.34 µm) around the immune synapse, ratio of intensity in the half of the cell near the synapse (front), and the half away from the synapse (back), for IIA1.6 cells treated with DMSO or SAHA in contact with a droplet for 15–20 min (median ± IQR, DMSO N = 23;16, SAHA N = 21;19, two independent experiments, Mann–Whitney test). ( E ) Western blot of GEF-H1 to evaluate the efficiency of GEF-H1 silencing. α-tubulin was used as a loading control. The blot presented is representative of two independent experiments. ( F ) Time-lapse images of F-actin in cells transfected with siCtrl or siGEF-H1 and treated with DMSO (control) or nocodazole, using SDCM 3D time-lapse imaging. Scale bar 5 µm. ( G ) Solidity in 2D and ( H ) aspect ratio of cells after 40  min of immune synapse formation (siCtrl DMSO N = 30;8, siCtrl Noco N = 19;4, siGEF-H1 DMSO N = 19;46, siGEF-H1 Noco N = 7;27, two independent experiments, Kruskal–Wallis test with multiple comparisons between DMSO and Noco, Dunn’s post test), analyzed on maximum z-projections of SDCM 3D images. ( I ) Left: examples of 3D SIM immunofluorescence imaging of F-actin and antigen on the droplet after 15–20 min of immune synapse formation. Side view: scale bar 5 µm. Front view: scale bar 2 µm. MIP visualization. Right: profiles of F-actin at the immune synapse, from symmetric radial scans of the immune synapse, normalized to the maxima (mean ± SEM, pooled from two experiments, siCtrl DMSO N = 11;7, siCtrl Noco N = 5;6, siGEF-H1 Noco N = 2;7). Figure 6—source data 1. Raw file of the full unedited Western blot images of , and a figure with annotated images of the full Western blot. Figure 6—source data 2. Data tables related to graphs in .

Article Snippet: Antibody , Biotin-SP-conjugated F(ab′) 2 goat polyclonal anti-mouse IgG , Jackson ImmunoResearch , 115-066-072 , Droplet functionalization (5.7 µL).

Techniques: Transfection, Immunofluorescence, Imaging, Confocal Microscopy, MANN-WHITNEY, Staining, Western Blot

Journal: eLife

Article Title: Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

doi: 10.7554/eLife.78330

Figure Lengend Snippet: Experiments for this figure were performed using F-tractin-tdTomato-expressing IIA1.6 cells in contact with a F(ab′) 2 αIgG-coated droplet and SDCM 3D time-lapse imaging. Cells were pretreated for 1 hr either with DMSO or with nocodazole 5 µM + para-nitroBlebbistatin 20 µM, kept in solution during the experiment. ( A ) Time-lapse images of F-actin-tdTomato-expressing cells, co-transfected with either a control empty vector (pRK5) or expressing RhoA CA (constitutively active). Scale bar 5 µm. ( B ) % coefficient of variation of 2D aspect ratio of individual cells over time, ( C ) Median 2D solidity of individual cells and ( D ) average distance of actin maxima to the droplet surface (median ± IQR, Control N = 10;9, RhoA CA N = 9;12, two independent experiments, Mann–Whitney test), analyzed on maximum z-projections. ( E ) Time-lapse images of F-tractin-tdTomato-expressing cells treated with DMSO or nocodazole + p-nBlebb, droplet outlined in blue. Scale bar 5 µm. ( F ) Median 2D solidity of maximum z-projections of individual cells over time (median ± IQR, DMSO N = 5;5;4, Noco + p-nBlebb N = 4;3;4, three independent experiments, Mann–Whitney test). ( G ) Aspect ratio of z-projections of cells in time (mean ± SEM, DMSO N = 5;5;4, Noco + p-nBlebb N = 4;3;4, three independent experiments). ( H ) Percentage of cells with aspect ratio >1.2 or <1.2 after 40 min of synapse formation. ( I ) Average distance of F-actin maxima to the droplet over 30 min of synapse formation (median ± IQR, DMSO N = 5;5;4, Noco + p-nBlebb N = 4;3;4, three independent experiments, Mann–Whitney test) (quantification: as in ). Figure 7—source data 1. Data tables related to graphs in .

Article Snippet: Antibody , Biotin-SP-conjugated F(ab′) 2 goat polyclonal anti-mouse IgG , Jackson ImmunoResearch , 115-066-072 , Droplet functionalization (5.7 µL).

Techniques: Expressing, Imaging, Transfection, Plasmid Preparation, MANN-WHITNEY

Journal: eLife

Article Title: Microtubules restrict F-actin polymerization to the immune synapse via GEF-H1 to maintain polarity in lymphocytes

doi: 10.7554/eLife.78330

Figure Lengend Snippet:

Article Snippet: Antibody , Biotin-SP-conjugated F(ab′) 2 goat polyclonal anti-mouse IgG , Jackson ImmunoResearch , 115-066-072 , Droplet functionalization (5.7 µL).

Techniques: Software, Sequencing, Cell Isolation, Transfection, Labeling, Injection, Imaging, Incubation, Recombinant