rabbit polyclonal primary anti cox 2 antibody  (Novus Biologicals)

Journal: bioRxiv

Article Title: Mapping Structure and Biological Functions within Mesenchymal Bodies using Microfluidics

doi: 10.1101/514158

Figure Lengend Snippet: (A-B) Schemes of the same MB where the cell centers are represented by black dots. Black lines show the Voronoi cells. The position of each cell in the MB is described by assigning a cell layer number (A, each color represent a cell layer, layer number 1 being the outermost cell layer) or by computing its normalized distance to the MB center r / R (B, r is the distance between the cell center and the MB center and R is the equivalent radius of the MB). (C-D) Evolution of the normalized cellular COX-2 signal with the cell layer number (C) and the normalized distance to the MB center (D). Black circles and errors bars represent respectively the mean normalized COX-2 signal and the standard deviation of the data. N chips = 13; n MBs = 2,936; n cells = 159,596. The layer analysis gives a much clearer trend with a higher COX-2 signal in the layers close to the MB edge, as can be seen in . The uncertainty of the cell center determination and the fact that two cells of a not perfectly round MB can have different r / R values even if they belong to the same concentric layer explain why the cell layer assignment is a more accurate determination of the cell location in the MB. ***: p < 0.001; N.S.: non significant.

Article Snippet: The samples were blocked with 5 % (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal anti-COX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1 % (v/v) FBS for 4 h. After washing with PBS, the samples were incubated with an Alexa Fluor ® 594 conjugate goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1 % (v/v) FBS, for 90 min.

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Mapping Structure and Biological Functions within Mesenchymal Bodies using Microfluidics

doi: 10.1101/514158

Figure Lengend Snippet: (A-B) Specificity of the staining and non-interference of the DMSO. Performing the immunostaining without the primary antibody and staining only with secondary antibody resulted in a very low fluorescent signal, which validated the specificity of the primary antibodies (A, control for secondary: N chips = 1; n MBs = 192; secondary: N chips = 1; n MBs = 251). MBs were formed and cultivated in the presence of 0.1 % (v/v) DMSO (the maximum concentration in culture media containing the inhibitors). Similar average per chip (A, control for DMSO: N chips = 3; n MBs = 644; DMSO 0.1 % (v/v): N chips = 1; n MBs = 195) and distribution of fluorescent signal in the cell layers (B, DMSO 0.1 % (v/v): n cells = 5,982) demonstrated the absence of contribution of 0.1 % (v/v) DMSO in the cell behavior within MBs. (C-D) Flow cytometry analysis of the percentage of COX-2 high (C) and N-cadherin (D) expressing cells, after MBs dissociation and immunostaining, revealed the presence of several subpopulations expressing different levels of these proteins, however, without any spatial information (at least 5000 cells were analysis for each condition). (E-F) Removing the blocking step during the staining showed that there is no limitation for antibody diffusion. The cells were fixed, permeabilized and then stained only with the secondary antibody, without blocking the samples (E), rendering all immunogenic sites of the MBs accessible. Fluorescent signal distribution in the different cell layers demonstrated higher signal intensity in the core of MBs than in the edge (F, Control: N chips = 13; n MBs = 2,936; n cells = 159,596; Secondary without blocking: N chips = 1; n MBs = 17; n cells = 1,618). (G-J) Quantifying the DAPI signal (G) and clearing the samples (H-J) showed that there was no significant light path alteration in the 3D MBs. (G) The DAPI fluorescent signal distribution inside the MBs displayed a continuous signal increase from the edge (r/R = 1) to the core (r/R = 0; N chips = 55; n MBs = 10,072; n cells = 699,836), which demonstrated that their in no diffusion limitation of small molecules and that the fluorescent light path is not attenuated by the MB opacity. (H-J) The MBs were subjected to ClearT2 treatment after the immunostaining for COX-2. Representative images (H) showed that the MBs were efficiently cleared post ClearT2, but the distribution of the fluorescent signal intensity was not affected as demonstrated by a representative MB (I) and the quantification of the distribution of the fluorescent signal after clearing in the different cell layers (J, control COX-2: N chips = 1; n MBs = 23; n cells = 2,366; clearing COX-2: N chips = 1; n MBs = 67; n cells = 6,333). The MBs were recovered from the chip then cryosectionned at 7 μm. For this cell layer depth, there is no antibody diffusion limitation or light path alteration. The COX-2 distribution signal show similar pattern as obtained with wide field imaging (K). Alternatively, the MBs were image using a 2-photons microscope and the COX-2 fluorescent signal pattern show similar distribution as wide field imaging (L). All scale bars are 50 μm. These results demonstrated the reliability of the measurements by image analysis, ensuring (1) the specificity of the fluorescent labeling; (2) the absence of limitation for antibody diffusion; (3) the absence of the light path alteration in the 3D structures.

Article Snippet: The samples were blocked with 5 % (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal anti-COX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1 % (v/v) FBS for 4 h. After washing with PBS, the samples were incubated with an Alexa Fluor ® 594 conjugate goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1 % (v/v) FBS, for 90 min.

Techniques: Staining, Immunostaining, Concentration Assay, Flow Cytometry, Expressing, Blocking Assay, Diffusion-based Assay, Imaging, Microscopy, Labeling

Journal: bioRxiv

Article Title: Mapping Structure and Biological Functions within Mesenchymal Bodies using Microfluidics

doi: 10.1101/514158

Figure Lengend Snippet: Evolution of the intra-MB fluorescent signal with control conditions (A-E, see in the main text) and inhibitors (F-I, see in the main text). (A) Normalized mean COX-2 with the cell layer number for MBs having different sizes (N chips = 13; blue, diameter < 110 μm, n MBs = 298, n cells = 9,282; purple, 145 μm< diameter < 165 μm, n MBs = 620, n cells = 42,469; red, diameter > 175 μm, n MBs = 295, n cells = 26,251) in control conditions. (B-E) Normalized mean COX-2 (B N chips = 13, n MBs = 2,936, n cells = 159,596), cadherin (C, with methanol fixation, N chips = 3, n MBs = 405, n cells = 24,185; D, with PFA fixation, N chips = 3, n MBs = 649, n cells = 47,254) and actin (E, N chips = 3, n MBs = 421, n cells = 23,970) signals with the cell layer number. Each color represents the mean behavior for one chip. (F-I) Evolution of the normalized mean COX-2 signal with the cell layer number depending on the inhibitor: QNZ (F, N chips = 6, n MBs = 1,215, n cells = 117,443), DAPT (G, N chips = 3, n MBs = 658, n cells = 37,165), Y27 (H, N chips = 4, n MBs = 709, n cells = 45,839) or CytoD (I, N chips = 3, n MBs = 458, n cells = 28,981). Each color represents the mean behavior for one chip. The black lines represent the mean of the single chips.

Article Snippet: The samples were blocked with 5 % (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal anti-COX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1 % (v/v) FBS for 4 h. After washing with PBS, the samples were incubated with an Alexa Fluor ® 594 conjugate goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1 % (v/v) FBS, for 90 min.

Techniques:

Journal: bioRxiv

Article Title: Mapping Structure and Biological Functions within Mesenchymal Bodies using Microfluidics

doi: 10.1101/514158

Figure Lengend Snippet: (A-B) RT-qPCR analysis of the relative TSG-6, COX-2, STC-1 and VEGF expression to GADPH (ΔCt, D and relative RNA expression, E), in 3D and in the 2D populations (n 3D =3, n 2D =3). (C) Quantification by ELISA of the PGE-2 and VEGF secreted by hMSCs cultivated in 2D, as MBs or as MBs treated with indomethacin (n chips = 3; n 2D = 3). (D) (D-E) Representative image (D) and quantitative analysis (E) of COX-2 (N chips = 13, n MBs = 2,936) and VEGF (N chips = 3, n MBs = 413) staining within the layers of the MBs, error bars represent the standard deviation) within the cell layers of the MBs. (E-H) of *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Article Snippet: The samples were blocked with 5 % (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal anti-COX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1 % (v/v) FBS for 4 h. After washing with PBS, the samples were incubated with an Alexa Fluor ® 594 conjugate goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1 % (v/v) FBS, for 90 min.

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Enzyme-linked Immunosorbent Assay, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Mapping Structure and Biological Functions within Mesenchymal Bodies using Microfluidics

doi: 10.1101/514158

Figure Lengend Snippet: (A) Representative images of MBs formed 1 day after the droplet loading, scale bar is 100 μm. Inhibitors are added to the culture medium prior to MB formation. (B-C) Quantitative analysis the aggregates projected area (B) and shape index (C) in the presence of the different inhibitors. Red lines represent the mean value for each condition. (D-E) Representative images (D, contrast is adjusted individually for a better visualization of the pattern, scale bar is 100 μm) and quantitative analysis (E) of the COX-2 fluorescent signal intensity normalized by the control value, with the different inhibitors. For these longer culturing times, QNZ and CytoD are only added during the phase change to allow MB formation. Small dots represent one MB. Large dots represent the average normalized COX-2 fluorescent signal per chip. Each color corresponds to a specific chip. *: p < 0.05; N.S. : non-significant. (F) Estimation of inhibitor effect in the cell layers with the COX-2 signal normalized by the control value. Control: N chips = 11, n MBs = 2,204; QNZ: N chips = 6, n MBs = 1,215; DAPT: N chips = 3, n MBs = 658; Y-27: N chips = 4, n MBs = 709; CytoD: N chips = 3, n MBs = 459. *: p < 0.05; **: p < 0.01; ***: p < 0.001; N.S.: non significant. (G) Proposed mechanisms regulating mesenchymal bodies formation and the patterning of their biological functions. (i) Regulation of the formation of mesenchymal bodies. (ii-iii) Spatial patterning of hMSC biological properties within mesenchymal bodies.

Article Snippet: The samples were blocked with 5 % (v/v) FBS in PBS for 30 min and incubated with a rabbit polyclonal anti-COX-2 primary antibody (ab15191, Abcam, Cambridge, UK) diluted at 1:100 in 1 % (v/v) FBS for 4 h. After washing with PBS, the samples were incubated with an Alexa Fluor ® 594 conjugate goat polyclonal anti-rabbit IgG secondary antibody (A-11012, Life Technologies, Saint Aubin, France) diluted at 1:100 in 1 % (v/v) FBS, for 90 min.

Techniques:

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

doi: 10.1186/s13018-017-0568-1

Figure Lengend Snippet: Real-time PCR analysis of CGRP and COX-2 mRNA expression in the infrapatellar fat pad and synovial tissue of knee OA patients. a CGRP and b COX-2 mRNA expression in the infrapatellar fat pad and synovial tissue of knee OA patients. *Statistically significant difference between IPFP and SYN ( p < 0.001). All data are presented as the mean ± standard error ( n = 100)

Article Snippet: The sections were reacted with primary mouse monoclonal primary antibody against CGRP (Abcam) and rabbit polyclonal primary antibody against COX-2 (Abcam, Cambridge, MA) for 6 h at 4 °C.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

doi: 10.1186/s13018-017-0568-1

Figure Lengend Snippet: Correlation between CGRP and COX-2 mRNA expression levels in the infrapatellar fat pad and SYN tissues of knee OA patients. Correlation between CGRP and COX-2 mRNA expression levels in IPFP tissues harvested from 99 knees of OA patients. The values for one IPFP were removed as outliers

Article Snippet: The sections were reacted with primary mouse monoclonal primary antibody against CGRP (Abcam) and rabbit polyclonal primary antibody against COX-2 (Abcam, Cambridge, MA) for 6 h at 4 °C.

Techniques: Expressing

Journal: Journal of Orthopaedic Surgery and Research

Article Title: Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

doi: 10.1186/s13018-017-0568-1

Figure Lengend Snippet: Relationship between CGRP and COX-2 mRNA expression level and K/L grade. a CGRP and b COX-2 mRNA expression in the infrapatellar fat pad of knee OA patients. All data are presented as the mean ± standard error

Article Snippet: The sections were reacted with primary mouse monoclonal primary antibody against CGRP (Abcam) and rabbit polyclonal primary antibody against COX-2 (Abcam, Cambridge, MA) for 6 h at 4 °C.

Techniques: Expressing