Dissected embryonic E14.5 brains were titrated in FACS buffer (PBS - 1% Fetal calf serum - 0.02% NaN3) using pipette tip, and filtered through 70-µm cell strainers (Fisher Scientific, Hampton, NH). Single cell suspensions were blocked with 10% normal rat serum on ice for 30 min., and stained with a combination of anti-mouse cell surface flow cytometric markers CD11b-FITC (BioLegend, San Diego, CA), GLAST-PE, and O4-APC (Miltenyi Biotec, Bergish Gladbach, Germany) at 4 °C in dark for 1 h with continuous rotation. The stained cells were then washed, centrifuged, and resuspended into 1 mL of FACS buffer, and acquired on a two-laser, six-color Gallios cytometer (Beckman Coulter, Pasadena, CA). The threshold for FACS analysis was set based on the surface expression of each individual marker in the samples as compared to isotype control using IgG-stained cells in the same channel, and Flow cytometric data were analyzed with the Kaluza 1.3 software (Beckman Coulter, Brea, CA), as reported [35 (link),36 (link)]. Cell populations were calculated as the percentages among the total cells.
Glast pe
Manufactured by Miltenyi Biotec
Sourced in United Kingdom, Germany
GLAST-PE is a fluorochrome-conjugated antibody that binds to the GLAST (glutamate aspartate transporter) protein. It is a useful tool for the identification and characterization of GLAST-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza 1, by Beckman Coulter (1 mentions)
Cd11b fitc, by BioLegend (1 mentions)
70 m cell strainer, by Thermo Fisher Scientific (1 mentions)
O4 apc, by Miltenyi Biotec (1 mentions)
Cd11b fitc, by Miltenyi Biotec (1 mentions)
Flow tube, by BD (1 mentions)
Cd45 pe cy7, by Miltenyi Biotec (1 mentions)
Kaluza, by Beckman Coulter (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cd11b apc vio770, by Miltenyi Biotec (1 mentions)
Facscanto 2, by BD (1 mentions)
Betulinic acid, by Bio-Techne (1 mentions)
Neun antibody, by Merck Group (1 mentions)
Real time pcr rt pcr primers, by Qiagen (1 mentions)
Antibodies against, by Fujifilm (1 mentions)
Antibodies against tgr5, by Abcam (1 mentions)
Phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Cd11b apc, by Thermo Fisher Scientific (1 mentions)
7 protocols using glast pe
1
Embryonic Brain Cell Isolation and Analysis
2
Multicolor Flow Cytometry for Glial Cell Analysis
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Mice (n=7+7 in both CUS and microinjection) were sacrificed with CO2. Tissues were gently homogenized through 70µm cell strainers (#352350, BD Biosciences) on ice as we described previously (24 (link), 32 (link)). Homogenates were blocked in PBS+10% rat serum for 1h with gentle rotation at 4°C. Fluorescent antibody makers (0.5µl/marker, mostly from Biolegend) diluted in 200µl PBS+1%FBS were added and incubated for 1h at 4°C with light protection. For CUS experiment, Plxnb2-PE (#145903), CD11b-BV421 (#101251), CD45-BV650 (#103151), and Glast-APC (#130-123-555) were used. For microinjection experiment, CD11b-FITC (#101206), CD45-PE/Cy7 (#103114), Glast-PE (#130-118-344, Miltenyi), and MHCII-BV711 (#107643) were used. Washed samples were finally resuspended with FC buffer and filtered through 35μm cell strainers into flow tubes (#08-771-23, BD Biosciences). The acquisition was made with BD LSR Fortessa™ (BD Biosciences). Data were analyzed using Kaluza (Beckman Coulter). Percentages (%) of positively stained cells were calculated.
3
Isolation and identification of microglia and astrocytes
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Microglia or astrocyte cultures were washed in sterile PBS, before scraping and gentle dissociation by pipetting. Cells were resuspended in FACS buffer (1% BSA/PBS) containing Fc Block (1 μg/mL). Cells were stained for CD11b‐APCVio770 and GLAST‐PE (Miltenyi Biotech, UK) for 30 min at 4°C. Cells were washed three times and resuspended in FACS buffer. Flow cytometeric data was collected using a FACSCanto II (BD Biosciences) cell analyzer. Microglia were identified by expression of CD11b and astrocytes by GLAST expression. Flow cytometeric analysis was carried out using FLOWJO version 7.6.5.
4
Immunohistochemical Analysis of Microglia
5
Immunoblotting and Flow Cytometry Techniques
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For immunoblotting: antibodies produced in rabbit against murine SBNO2, C‐terminal peptide (1:640), were custom prepared by Biomatik (Cambridge, ON, Canada). Antibodies against STAT3 (1:1,000), phospho‐STAT3 (Tyr705, 1:2,000), phospho‐p44/42 MAPK ((phospho)‐ERK 1/2) (Thr 202/Tyr 204, 1:3,000), NFκB (p65, 1:3,000), phospho‐NFκB (p65, Ser536, 1:1,000) all produced in rabbit were from Cell Signaling. The ERK 1/2 antibody (1:10,000, host: rabbit) was from Sigma‐Aldrich, while GAPDH antibody (1:100,000, host: mouse) was from Millipore. Horse raddish peroxidase (HRP)‐coupled goat anti‐rabbit IgG (SC2004, Santa Cruz Biotechnology Inc., Dallas, TX) or goat anti‐mouse IgG (Fc specific, A0168, Sigma‐Aldrich) secondary antibodies were used for detection.
For flow cytometry: CD11b‐APC (1:200, eBioscience, San Diego, CA) and GLAST‐PE (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies plus corresponding isotype controls were used.
For flow cytometry: CD11b‐APC (1:200, eBioscience, San Diego, CA) and GLAST‐PE (1:11, Miltenyi Biotec, Bergisch Gladbach, Germany) antibodies plus corresponding isotype controls were used.
6
Characterizing Adult Subependymal Zone Cells
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Characterization of NSC and NPC populations in the adult SEZ was performed as previously described [38 (link), 39 (link)]. Roughly, SEZ tissue was dissected, minced and enzymatically digested using the neural tissue dissociation kit (T) (Miltenyi, 130–093-231) in a gentleMACS Octo Dissociator with heaters (Miltenyi). After trypsin inhibition, digested pieces were mechanically dissociated, the cell suspension was filtered through a 40 μm and treated with the Dead Cell Removal Kit (Miltenyi, cat no. 130-090-101) following the instructions of the manufacturer. Finally, the eluted living fraction was pelleted (300×g, 10 min) and incubated with the specific cocktail of primary antibodies [38 (link), 39 (link)] (1:300 CD24-PerCP-Cy5.5, BD 562360; 1:100 CD31-BUV395, BD 740239; 1:200 CD45-BUV395, BD 565967; 1:20 CD9-Vio770, Miltenyi 130-102-384; 1:20 GLAST-PE, Miltenyi 130-095-821; 1:30 O4-Biotin, Miltenyi 130-095-895; 1:200 Ter119-BUV395, BD 563827; 1:300 AF488 EGF complex, Molecular Probes E13345) and reagents (DAPI, 50 µg/ml) at 4 °C for 30 min. Labeled samples were analyzed using a LSR-Fortessa cytometer (Becton Dickinson) with 350, 488, 561 and 640 nm lasers.
7
Extracellular Vesicle Capture and Analysis
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The bead/antibody/EV complex was (a) coupled to a fluorescein isothiocyanate (FITC) fluorescent tag that binds to extracellular vesicles (Exo-FITC, Systems Biosciences; Cat #CSFLOWBASICA-1) and a phycoerythrin (PE) fluorescent tag that specifically binds to the astrocyte surface marker, GLAST-PE (Miltenyi Biotec Inc; Cat #130-118-483); and (b) subsequently analyzed by flow cytometry to confirm AEEV capture. The bead/antibody/EV complex was washed three times with 1X BWB and then incubated with 10 μL of Exo-FITC and 2 μL of GLAST-PE in 240 μL of Exosome Stain Buffer for 2 h on ice with gentle flicking every 30 min. The stained complex was washed three times in 1X BWB and resuspended in 500 μL 1X BWB prior to flow cytometry analysis. The flow cytometric data were acquired using a BD LSR II Special Order Flow Cytometer (BD Biosciences, San Jose, CA). Instrument performance was validated using BDTM Cytometer Setup and Tracking (CS&T) beads (BD Biosciences, San Jose, CA). All data were analyzed using FACS DIVA 8.0 software (BD Biosciences). Debris and small particles were excluded by gating out events with low forward scatter. Fig. 1 B shows an example of successful EV capture, and Fig. 1 C shows an example of AEEV enrichment.
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