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Glast apc

Manufactured by Miltenyi Biotec
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Sourced in Germany, United States, Spain

GLAST-APC is a fluorescently labeled antibody product designed for flow cytometric analysis. It targets the GLAST (Glutamate Aspartate Transporter) antigen, which is expressed on cells. The APC (Allophycocyanin) fluorophore is conjugated to the antibody, allowing for detection of GLAST-positive cells.

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Lab products found in correlation

Kaluza v2, by Beckman Coulter (2 mentions) Cd11b bv421, by BioLegend (2 mentions) Fortessa flow cytometer, by BD (2 mentions) O4 pe, by Miltenyi Biotec (2 mentions) Cd45 bv650, by BioLegend (2 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions) Cd45 fitc, by Miltenyi Biotec (2 mentions) Vwf fitc, by Abcam (2 mentions) Dnase 1, by Merck Group (1 mentions) Collagenase type 4, by Merck Group (1 mentions) Cd45 bv421, by BioLegend (1 mentions) Facsymphony s6 cell sorter, by BD (1 mentions) Cd49f percp cy5, by BioLegend (1 mentions) Igm pe, by BioLegend (1 mentions) 70 μm cell strainer, by BD (1 mentions) Facsaria fusion cell sorter, by BD (1 mentions) Stempro accutase, by Thermo Fisher Scientific (1 mentions) Dnase 1, by Qiagen (1 mentions) Fortessa, by BD (1 mentions) Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

GLAST-1-APC (2 citations) Anti-GLAST-APC (2 citations)

6 protocols using glast apc

1

Glial Cell Profiling in Mouse Brain

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Hippocampal homogenates were washed, centrifuged at 500 g for 5 min, and blocked with PBS + 10% rat serum for 1 h, then stained with flow markers (BioLegend and Miltenyi) of anti-mouse Csf1r-Brilliant Violet (BV)605 (135517, BioLegend), CD11b-BV421 (101251, BioLegend), CD45-BV650 (103151, BioLegend), Glast-APC (130–123-555, Miltenyi), and O4-PE (130–117-357, Miltenyi) for 1 h. Washed cells were resuspended in 500 µl PBS and acquired with a Fortessa flow cytometer (BD Bioscience). Data were analyzed by Kaluza v2.1 software (Beckman Coulter). Astrocytes were defined as Glast+ cells, oligodendrocyte precursor cells (OPCs) as O4+ cells, microglia as CD45lowCD11bhi cells. The % of glia among total brain cells and mean fluorescent intensity (MFI) of Csf1r per microglia were measured and total Csf1r protein level was calculated as MFI x microglial No.
Yan L., Li Y., Fan F., Gou M., Xuan F., Feng W., Chithanathan K., Li W., Huang J., Li H., Chen W., Tian B., Wang Z., Tan S., Zharkovsky A., Hong L.E., Tan Y, & Tian L. (2023). CSF1R regulates schizophrenia-related stress response and vascular association of microglia/macrophages. BMC Medicine, 21, 286.
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2

Multiparametric Cell Sorting of Cerebrovascular Cells

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Tissue was cut and minced on ice, and then enzymatically digested twice with 1 mg/ml Collagenase Type IV (MilliporeSigma, Darmstadt, Germany) and 100 μg/ml DNase I (MilliporeSigma) at 37℃ for 20 min and 30 min, respectively. The cell suspension was filtered, washed, and pelleted. Cells were then resuspended with 25% Percoll and underwent 20 min centrifugation without break. The top layer containing cell debris and myelin was removed. A multispectral LED light was used to perform a 30-min irradiation treatment to reduce background autofluorescence [23 ]. Cells were stained with an anti-human CD31-PE (303,105, BioLegend, San Diego, United States), CD45-BV421 (304,031, BioLegend), CD13-PE-Cy7 (301,711, BioLegend), P2RY12-FITC (392,107, BioLegend), CD49f-PerCP-Cy5.5 (313,617, BioLegend), CD90-BV711 (328,139, BioLegend) and GLAST-APC (130–123-555, Miltenyi Biotec, Bergisch Gladbach, Germany) antibody cocktail. Size, granularity, and antibody-specific gating were set to sort ECs, pericytes, microglia and neuroglia using the FACSymphony S6 Cell Sorter (BD Biosciences, Franklin Lakes, United States) (Fig. S1a).
Li Y., Girard R., Srinath A., Cruz D.V., Ciszewski C., Chen C., Lightle R., Romanos S., Sone J.Y., Moore T., DeBiasse D., Stadnik A., Lee J.J., Shenkar R., Koskimäki J., Lopez-Ramirez M.A., Marchuk D.A., Ginsberg M.H., Kahn M.L., Shi C, & Awad I.A. (2024). Transcriptomic signatures of individual cell types in cerebral cavernous malformation. Cell Communication and Signaling : CCS, 22, 23.
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3

Quantification of Brain Cell Populations

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Mouse hippocampi were dissected after CO2 euthanization and gently homogenized through 70 μm cell strainers (#352350, BD Bioscience, United States) in ice-cold PBS with 1% FBS. Homogenates were washed and centrifuged at 500 g for 5 min. Isolated cells were blocked with 10% rat serum in ice-cold PBS for 1 h. Brain cells were stained with 0.5 μL anti-mouse VGLUT2-Alexa488 (#MAB5504A4, Millipore, United States), CD11b-BV421 (#101251, BioLegend, United States), CD45-BV650 (#103151, BioLegend, United States), Glast-APC (#130–123-555, Miltenyi, Germany), and O4-PE (#130–117-357, Miltenyi, Germany) in PBS with 1% FBS on ice for 1 h. Corresponding isotype control antibodies (all BioLegend, United States) included rat IgG2a-Alexa488 (##400,525), IgG2b-BV421 (#400639), IgG2b-BV650 (#400651), IgG2b-APC (#400219), and IgM-PE (#401611). Cells were washed, resuspended in 500 mL PBS, and acquired with a Fortessa flow cytometer (BD Bioscience, United States). Data were analyzed by Kaluza v2.1 software (Beckman Coulter, United States). Astrocytes were defined as Glast+ cells, oligodendrocyte precursor cells (OPCs) as O4+ cells, and microglia as CD45lowCD11bhi cells. Cell populations were calculated as % among total brain cells or microglia as previously described (Piirainen et al., 2021 (link); Chithanathan et al., 2022 (link)).
Yan L., Xuan F.L., Chen S., Gou M., Chen W., Li Y., Wang Z., Wang L., Xie T., Fan F., Zharkovsky A., Tan Y, & Tian L. (2023). Replenished microglia partially rescue schizophrenia-related stress response. Frontiers in Cellular Neuroscience, 17, 1254923.
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4

Astrocyte Enrichment and Purification

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Day 60 iAstros were washed with HBSS without Mg Ca (Gibco) three times then lifted using a 10–15 minute incubation of StemPro Accutase (Gibco) at 37°C. Cells were collected and strained through a 70 μm cell strainer then incubated with 100U/mL DNaseI (Qiagen) in FACS Buffer (0.5% BSA [Gibco] in PBS without Mg and Ca) for 10 minutes at room temperature. Cells were washed, collected, then stained for GLAST-APC (Miltenyi Biotec; see Table S3) in FACS Buffer for 15 minutes at 4°C in the dark. Cells were washed, 40 μm strained, then collected, then stained for DAPI in FACS Buffer for 5 minutes at room temperature in the dark. Cells were immediately sorted on the FACSAria Fusion Cell Sorter (BD Biosciences) into ADM3 with PenStrep (Gibco). GLAST-positive/DAPI-negative cells were replated on hESC-qualified matrigel at a seeding density of 4e4 cells/cm2 (0.38e6 cells/well of a 6-well) in ADM3 with 1x PenStrep. Following astrocyte-enrichment, the cells were cultured for another 7 days before harvest with half media changes every other day in ADM3 with 1x PenStrep followed by immunocytochemistry quality control analysis for astrocyte markers.
Reyes-Ortiz A.M., Abud E.M., Burns M.S., Wu J., Hernandez S.J., McClure N., Wang K.Q., Schulz C.J., Miramontes R., Lau A., Michael N., Miyoshi E., Van Vactor D., Reidling J.C., Blurton-Jones M., Swarup V., Poon W.W., Lim R.G, & Thompson L.M. (2022). Single-nuclei transcriptome analysis of Huntington disease iPSC and mouse astrocytes implicates maturation and functional deficits. iScience, 26(1), 105732.
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5

Mitochondria and Cell Surface Marker Analysis

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Standard FACS analysis was performed by BD Fortessa as described before 10 (link). To label mitochondria, MitoTracker Deep Red (100 nM, Thermo Fisher Scientific) or MitoTracker Red CMXRos (100 nM, Thermo Fisher Scientific) was used. Each fluorescent conjugated antibody such as vWF-FITC (1:500, Abcam), GLAST-APC (1:500, Miltenyl Biotec), CD45-FITC (1:500, Miltenyl Biotec), or CD41/CD61-FITC (1:500, Miltenyl Biotec) was added in CSF sample (100 μL) for 1 hour at room temperature. FACS analysis was performed using an unstained or phenotype control for determining appropriate gates, voltages, and compensations required in multivariate flow cytometry.
Chou S.H., Lan J., Esposito E., Ning M., Balaj L., Ji X., Lo E.H, & Hayakawa K. (2017). Extracellular mitochondria in cerebrospinal fluid and neurological recovery after subarachnoid hemorrhage. Stroke, 48(8), 2231-2237.
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6

Extracellular Mitochondria Identification in CSF

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Fluorescence-activated cell sorter analysis (FACS) was performed to identify extracellular mitochondria in the CSF samples (BD FACSCalibur, Becton–Dickinson, San Jose, CA, USA), according to our previous reports17 (link),18 (link). A 100-µl CSF sample was stained with 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, Waltham, MA, USA) for 40 min at room temperature. The potential cellular origin of the extracellular mitochondrial signals was evaluated using the following fluorescence-conjugated antibodies: von Willebrand factor conjugated with fluorescein isothiocyanate (vWF-FITC; Abcam, Cambridge, MA, USA), glutamate aspartate transporter conjugated with allophycocyanin (GLAST-APC, Miltenyl Biotec, Madrid, Spain), CD45-FITC (Miltenyl Biotec), and CD41/61-FITC (Miltenyi Biotec) (Supplementary Table S1). The CSF samples were immunostained with the antibodies for 20 min at room temperature and analyzed by flow cytometry. The data were analyzed using Flow Jo software (v 10.7.1 Ashland, OR, USA)17 (link),18 (link).
Youn D.H., Kim N., Lee A., Han S.W., Kim J.T., Hong E.P., Jung H., Jeong M.S., Cho S.M, & Jeon J.P. (2023). Autophagy and mitophagy-related extracellular mitochondrial dysfunction of cerebrospinal fluid cells in patients with hemorrhagic moyamoya disease. Scientific Reports, 13, 13753.
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