Colonies were dissociated with Accutase and rinsed twice with 1× PBS with 0.5% (w/v) BSA. Cells were counted using a hemocytometer, and roughly 1 × 106 cells per sample were used for analysis. All cells were fixed with 4% (v/v) paraformaldehyde for 40 min at 4 °C, washed twice with PBS, permeabilized using 0.1% (v/v) Triton X-100 in PBS, and blocked using 0.5% (w/v) BSA in PBS. Cells were stained with conjugated antibodies for 20 min in the dark on ice. Primary conjugated antibodies were the following: MAP2-PE (FCMAB318PE, Millipore), TUJ1 Alexa Fluor 488 (560381, BD Pharmingen), MAP2B Alexa Fluor 647 (560382, BD Pharmingen), OCT3/4 Alexa Fluor 647 (560329, BD Pharmingen), and SOX2 Alexa Fluor 488 (561593, BD Pharmingen). Flow cytometry (BD FACs Calibur from BD Biosciences) was performed, and data were analyzed using FlowJo software v9.8.1 (TreeStar Inc.). Total cell population was identified and gated based on characteristic voltage pulse area measurement of forward scatter (FSC: particle size) and side scatter (SSC: granularity/complexity of the particle) of the laser light.
Fcmab318pe
Manufactured by Merck Group
Sourced in United States
FCMAB318PE is a lab equipment product manufactured by Merck Group. It is a fluorescent-conjugated monoclonal antibody designed for use in flow cytometry analysis. The core function of this product is to facilitate the detection and quantification of specific target molecules or cells in a sample.
Automatically generated - may contain errors
2 protocols using fcmab318pe
1
Characterization of Neuronal Cell Populations
2
Multiparametric Flow Cytometry Analysis
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APC‐conjugated anti‐CD44 (#103011, 1:200; Biolegend, San Diego, CA, USA), phycoerythrin (PE)‐conjugated anti‐glial fibrillary acidic protein (GFAP; #FCMAB257P, 1:40; Millipore, Burlington, MA, USA), PE‐conjugated anti‐microtubule‐associated protein 2 (MAP2; #FCMAB318PE, 20 μl per test; Millipore), and control IgG (#555749, 1:40; BD Biosciences) antibodies were used for FACS. To obtain CD44High or CD44Low cells, patient‐derived primary tumor cells or transplanted murine primary tumor cells stained with 1:200 dilutions of APC‐conjugated anti‐CD44 antibody were subjected to FACS. For cell cycle analyses, cells were incubated with 20 μm BrdU (#B5002; Sigma‐Aldrich, St Louis, MO, USA) for 3 days, followed by incubation with PE‐conjugated anti‐BrdU antibody (#339811, 1:40; Biolegend). Apoptotic cell death was measured using an APC‐ (#ab236215; Abcam, Cambridge, UK) or FITC‐conjugated (#ab14085; Abcam) Annexin V Apoptosis Detection Kit I.
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