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4 protocols using lngfr pe

1

Immunophenotyping of Cultured Cells

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Digested cells were suspended in HBSS at a density of 1–5 × 107 cells/mL and stained for 30 minutes on ice with THY-1-APC (BD Pharmingen) and LNGFR-PE (Miltenyi Biotec) antibodies for sorting. Cultured cells (at three and five passages) were harvested using cell-dissociation buffer (Gibco). Cells (1.0 × 105) were suspended in ice-cold HBSS and stained for 30 minutes on ice with the monoclonal antibodies CD45-PE-Cy7 (Tonbo Biosciences), CD29-PE, CD31-PE-Cy7, CD44-PE, CD105-PE and CD166-PE (BioLegend) for cell surface analysis. Flow cytometric analysis and cell sorting were performed on a triple-laser Moflo system (Beckman Coulter) and data were analyzed using Flowjo software (Tree Star).
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2

Multiparametric Flow Cytometry Analysis

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Data were acquired on a MACSQuant® Analyzer 10 or MACSQuant® X (Miltenyi Biotec) and analyzed with FlowLogic™ V.8 (Inivai Technologies) by either frequency or median fluorescence intensity (MFI). Cells were stained in CliniMACS® PBS/EDTA Buffer (Miltenyi Biotec, catalog no.: 200-070-025) with 0.5% BSA at 4°C for 10 min. Antibodies used for flow cytometry: CD137-APC (clone: REA765, Miltenyi Biotec), CD25-PE-Vio770 (clone: REA570, Miltenyi Biotec), CD69-VioBlue (clone: REA824, Miltenyi Biotec), LNGFR-PE (clone: REA844, Miltenyi Biotec), CD4-VioGreen (clone: REA623), CD8-APC-Vio770 (clone: REA734, Miltenyi Biotec), CD3-FITC (clone: REA613, Miltenyi Biotec), CD45-VioBlue (clone: REA747, Miltenyi Biotec), CD279-PE-Vio770 (clone: REA1165, Miltenyi Biotec), CD366-APC (clone: REA635, Miltenyi Biotec), CD223-FITC (clone: REA351, Miltenyi Biotec). Antibodies were used according to the manufacturer’s protocol.
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3

CAR T Cell Stimulation Assay

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A total of 3 × 105 irradiated NIH/3T3 cells expressing human CD19 were plated on 24-well plates and used to stimulate 1 × 106 CAR T cells per ml as previously described12 (link). Cells were maintained in RPMI-1640 medium supplemented with 10% FBS without addition of cytokines. For in vitro assays, CAR T cells were bead-sorted four days after T cell transduction and before co-culture with NIH/3T3 cells. CAR T cells were stained with LNGFR–PE (clone C40-1457, BD antibody followed by co-incubation with anti-PE beads (Miltenyi). All in vitro assays were performed 7 days after stimulation on CD19+ NIH/3T3 cells. T cells were enumerated using an automated cell counter (Nexcelom Bioscience).
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4

Isolation and Sorting of Human Bone Marrow Cells

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All BM‐MNC experiments were performed using Poietics BM‐MNCs purchased from LONZA. Ethical approval for the generation of these cells was comprehensively undertaken by LONZA. hMSCs were prepared from human BM (human BM‐MNCs; Lonza, Amagazaki, Japan) as described in our previous report and in the Supplemental Experimental Procedures.30, 35 All experimental protocols were approved by the animal committee of Keio University, Japan. All methods were conducted in strict accordance with the approved guidelines of the institutional animal care committee. LNGFR‐PE (Miltenyi Biotec, Bergisch‐Gladbach, Germany) and THY‐1‐APC (BD Pharmingen, San Jose, California) were added to the human BM cells and incubated for 30 minutes on ice. The tube was centrifuged, the supernatant was discarded, and HBSS containing propidium iodide (Sigma, St. Louis, Missouri) was added. The cells were sorted using a triple‐laser MoFlo (Beckman Coulter, Brea, California), FACS Vantage SE, or FACS Aria III (Becton Dickinson, Bedford, Massachusetts). The data were analyzed using FlowJo software (Tree Star, Ashland, Oregon). All flow cytometry experiments are described in the Supplemental Experimental Procedures.
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