At the end of the expansion phase in the 3D culture system, cells were extracted by substituting the CM with a solution of 0.3% collagenase (collagenase) and perfusing the ceramic constructs for 40 min followed by 0.05% trypsin/0.53 mM EDTA solution (trypsin) for additional 15 min both at 400 µm per second. Extracted cells were subsequently sorted using anti-CD45-coated magnetic beads (Miltenyi Biotec, Auburn, CA), according to the manufacturer's instructions. 2D-expanded cells were retrieved by using the same enzymatic solutions, i.e. collagenase for 40 min and trypsin for 5 min. The fraction of dead cells, preliminarily assessed by assessed by Trypan blue exclusion (Sigma, Switzerland), was negligible (less than 3%), with no obvious differences between the experimental groups. Both CD45+ and CD45− viable cell populations were assessed for the ability to form fibroblastic colonies. The CD45− populations were further characterized by flow cytometry, gene expression by means of microarray analysis and quantitative real-time (QRT) PCR or tested for the multilineage differentiation capacity, as described below.
Anti cd45 coated magnetic beads
Manufactured by Miltenyi Biotec
Sourced in Germany
Anti-CD45-coated magnetic beads are a laboratory product used for the isolation and purification of CD45-positive cells from various sample types. The beads are coated with antibodies specific to the CD45 surface antigen, allowing for the selective capture and separation of CD45-expressing cells from a heterogeneous cell population.
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2 protocols using anti cd45 coated magnetic beads
1
Isolation and Characterization of CD45+ and CD45- Cells
2
Isolation and Characterization of Antigen-Presenting Cells
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Antigen‐presenting cells were obtained following the protocol described by Tanaka et al.20 Briefly, the mouse sublingual mucosa tissues were excised by trimming off the roots of the tongue and cut into fragments. Tissues were treated with 0.25% trypsin and 1 mM EDTA for 1 hours at 37⁰C with shaking. Next, they were incubated (1 hours at 37⁰C with shaking) with 1 mg/mL collagenase (Wako Pure Chemical) and 0.1 mg/mL DNase I (Sigma‐Aldrich) in RPMI medium containing 10% fetal bovine serum. Cells were then filtered, using a 70‐mm cell strainer. After washing, cells were purified by density gradient centrifugation with Ficoll‐Histopaque (VWR) following a standard protocol. CD45 population was isolated with anti‐CD45‐coated magnetic beads according to the supplier′s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells obtained were characterized phenotypically by flow cytometry on an FC‐500 Beckman Coulter using FLOWJO V.10 software. Briefly, 105 cells were stained (30 minutes at 4°C) with anti‐CD45 (mAb clone 104), anti‐MHC class‐II (mAb clone M5/114.15.2), anti‐CD207 (mAb clone 4C7), anti‐CD11b (mAb clone M1/70), and anti‐CD64 (mAb clone X54‐5/7.1) (Biolegend). The corresponding isotype controls were included in each staining.
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