Journal: Biochemistry and Biophysics Reports

Article Title: Ligand-competent fractalkine receptor is expressed on exosomes

doi: 10.1016/j.bbrep.2021.100932

Figure Lengend Snippet: Expression of tetraspanins on exosomes isolated from mouse cells. The exosomes were isolated from the culture media, immobilized on microbeads, and stained with the indicated monoclonal antibody (MAb) (see Materials and Methods for details). The tetraspanin expressions were analyzed by using flow cytometry for the exosomes of mouse T cells or T-cell lines ( A ), mouse cancer cell lines (B16F10, CT26.WT, and LTPA) ( B ), mouse macrophage (RAW264.7) and bone (MLO-Y4 and MLO-A5) cell lines ( C ), and mouse primary cells isolated from spleens, bone marrows, and lungs ( D ). Flow cytometry histograms show the expression of the indicated markers. Data are representative of three separate analyses. Red lines, MAb; and black lines, isotype. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MLO-Y4 (osteocyte) and MLO-A5 (osteoblast) cells were purchased from Kerafast (Boston, MA, USA).

Techniques: Expressing, Isolation, Staining, Flow Cytometry

Journal: Biochemistry and Biophysics Reports

Article Title: Ligand-competent fractalkine receptor is expressed on exosomes

doi: 10.1016/j.bbrep.2021.100932

Figure Lengend Snippet: Expression of CCR9, CXCR4, and CX3CR1 on the exosomes of various cell types. The exosomes isolated from the culture media of various cell types after being cultured for 48 h were immobilized on microbeads and stained with the indicated monoclonal antibody (MAb) (see Materials and Methods for details). The expression levels of those chemokine receptors on the exosomes of several mouse cell lines (B16F10, CT26.WT, LTPA, MLO-Y4 and MLO-A5) ( A ) and mouse primary cells isolated from spleen, bone marrow, and lung ( B ) were measured by using flow cytometry. The exosomes were immobilized on microbeads, stained with the indicated MAb, subjected to flow cytometry and analyzed for the expression of those chemokine receptors. CCR9, C–C chemokine receptor type 9; CXCR4, C-X-C chemokine receptor type 4; and CX3CR1, C-X3-C motif chemokin receptor 1. Flow cytometry histograms show the expression of the indicated chemokine receptors. Data are representative of three separate analyses. Red lines, MAb; and black lines, isotype. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MLO-Y4 (osteocyte) and MLO-A5 (osteoblast) cells were purchased from Kerafast (Boston, MA, USA).

Techniques: Expressing, Isolation, Cell Culture, Staining, Flow Cytometry

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone

doi: 10.1002/jor.23665

Figure Lengend Snippet: A–B) MLO-Y4 osteocytes were loaded with various magnitudes of fluid flow shear stress for 2 hours using medium containing 5% FBS, 5% BCS, 1% penicillin/streptomycin, and 1 mg/mL fluorescein-conjugated dextran. PMD-labeled osteocytes (green) were quantified from randomly collected confocal microscopy images (n=10 to 12 images per condition) after rinsing samples thoroughly in PBS to remove residual dye. *p≤0.05 vs. control for each experiment; each control/flow pairing is representative of three independent experiments. C–D) A 20 dynes/cm2 (2 Pa) shear stress was applied to MLO-Y4 osteocytes either immediately (1 step) or by gradually ramping up to this load (2 to 13 steps); PMD affected osteocytes were quantified as in panel B. *p≤0.05 vs. 1-step; representative of two independent experiments. E–F) Experiments in panels B and D were repeated with MLO-Y4 cells using medium containing 1% FBS, 1% FBS, 1% penicillin/streptomycin, 1 mg/mL fluorescein-conjugated dextran, and 1 mg/mL bovine serum albumin. *p≤0.05 vs. control or 1-step, representative of four independent experiments.

Article Snippet: Fluid flow shear stress: Glycotech flow chamber MLO-Y4 cells were seeded 4 days prior to experiments onto type 1 collagen coated dishes; cells were 70% confluent at the time of experiments.

Techniques: Shear, Labeling, Confocal Microscopy, Control

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone

doi: 10.1002/jor.23665

Figure Lengend Snippet: A) MLO-Y4 osteocytes were subjected to fluid flow shear stress (30 dynes/cm2) for 5 minutes using culture medium (5% FBS, 5% BCS, 1% penicillin/streptomycin) containing 1 mg/mL fluorescein-conjugated dextran. Cells were imaged in real-time during flow, which confirmed the absence of air bubbles during testing. PMD-labeled osteocytes (green) were observed within the flow path after rinsing samples thoroughly in PBS to remove residual dye. B) PMD-labeled osteocytes (green) from experiments in panel A were quantified from randomly collected confocal microscopy images within the flow path (n=5 images per condition) after rinsing samples thoroughly in PBS to remove residual dye. *p≤0.05 vs. control; data represent mean ± SEM of five independent experiments. C)MLO-Y4 osteocytes were subjected to fluid flow shear stress (40 dynes/cm2) using culture medium containing 2 μM FM4-64 dye, which permitted observation of membrane uptake of dye (white arrows) in real-time during flow loading. Each control/flow pairing is representative of three independent experiments.

Article Snippet: Fluid flow shear stress: Glycotech flow chamber MLO-Y4 cells were seeded 4 days prior to experiments onto type 1 collagen coated dishes; cells were 70% confluent at the time of experiments.

Techniques: Shear, Labeling, Confocal Microscopy, Control, Membrane