Acsa 2 pe

Manufactured by Miltenyi Biotec

Sourced in Spain

ACSA-2-PE is a fluorescent-labeled reagent for the detection of activated caspase-2 in cells. It binds specifically to activated caspase-2 and can be used for flow cytometric analysis.

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Lab products found in correlation

O4 apc, by Miltenyi Biotec (3 mentions) Adult brain dissociation kit, by Miltenyi Biotec (2 mentions) Facscanto 2, by BD (2 mentions) Cd11b pe cy7, by Thermo Fisher Scientific (2 mentions) Facsverse cytometer, by FlowJo (1 mentions) Cd11b vio bright fitc, by Miltenyi Biotec (1 mentions) Fcr blocking reagent, by Miltenyi Biotec (1 mentions) Mouse fcγ receptor block reagent, by BD (1 mentions) Apc cy7 rat anti cd11b clone m1 70, by BD (1 mentions) Brilliant stain buffer, by BD (1 mentions) Neuron isolation kit, by Miltenyi Biotec (1 mentions) Gentlemacs octo dissociator with heater, by Miltenyi Biotec (1 mentions) Cd45 fitc, by BioLegend (1 mentions) Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Ly6g apc cy7, by BioLegend (1 mentions) Cd11b bv421, by BD (1 mentions) Fam flica reagent, by ImmunoChemistry Technologies (1 mentions) Cd11c apc, by BioLegend (1 mentions) Cd45 percp cy5, by BD (1 mentions)

4 protocols using acsa 2 pe

Astrocyte Purity Validation by Flow Cytometry

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The purity of the astrocyte cultures was confirmed by flow cytometry. First, cells were blocked with FcR blocking reagent (#130-092-575, Miltenyi Biotec, Madrid, Spain,) in 1× phosphate buffer saline (PBS) and 0.5% bovine serum albumin (BSA) and then stained with combinations of fluorophore-conjugated antibodies purchased from Miltenyi Biotec: for astrocytes, ACSA-2-PE (#130-102-365); for microglia, CD11b-Vio Bright FITC (#130-109-368); and for oligodendrocytes, O4-APC (#130-095-895). Data were acquired on a FACSVERSE cytometer and analyzed using FlowJo v10 software (Ashland, OR, USA). Non-viable cells were excluded based on forward and side scatter profiles and 7-Aminodactinomycin (7AAD) staining (#130-111-568).

Moreno-Estellés M., Campos-Rodríguez Á., Rubio-Villena C., Kumarasinghe L., Garcia-Gimeno M.A, & Sanz P. (2023). Deciphering the Polyglucosan Accumulation Present in Lafora Disease Using an Astrocytic Cellular Model. International Journal of Molecular Sciences, 24(7), 6020.

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Single-Cell Dissociation and Sorting

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Mouse brains were dissociated into single cell suspension using the Adult Brain Dissociation Kit (Miltenyi Biotec, Cat# 130-107-677) with cell debris removal following manufacturer’s instructions. Single cells were resuspended in cold MACS buffer supplemented with mouse Fcγ receptor block reagent (553141; BD Biosciences) and incubated on ice for 5 min, followed by staining with fluorescently tagged antibodies in the brilliant stain buffer (563794; BD Biosciences) on ice for 20min. The cells were washed twice with MACS buffer, and used for FACS sorting. Antibodies: APC-Cy7 rat anti-CD11b (clone M1/70, Cat# 557657; BD Biosciences), ACSA2-PE (Miltenyi Biotec, Cat# 130-123-284), O4-APC (Miltenyi Biotec, Cat# 130-118-978), BV421 Rat Anti-Mouse CD24 (clone M1/69, Cat# 562563; BD Biosciences).

Shi Y., Andhey P.S., Ising C., Wang K., Snipes L.L., Boyer K., Lawson S., Yamada K., Qin W., Manis M., Serrano J.R., Benitez B.A., Schmidt R.E., Artyomov M., Ulrich J.D, & Holtzman D.M. (2021). Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms. Neuron, 109(15), 2413-2426.e7.

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Neuronal Isolation from Adult Mouse Brains

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Whole brains from adult female 10- to 15-wk-old mice were dissociated using the Adult Brain Dissociation Kit and Gentle Macs Octo Dissociator with Heaters (Miltenyi) according to the manufacturer’s protocol. Non-neuronal cells were removed using the Neuron Isolation Kit (Miltenyi), which contains biotinylated antibodies against microglia, oligodendrocytes, and astrocytes, conjugated to magnetic microbeads that are removed by magnetic-activated cell sorting (MACS). The flow-through containing highly enriched neuronal cells was collected. The purity of neuronal isolates was assessed by flow cytometry using a FACS Canto II (BD Biosciences) according to the manufacturer’s instructions. In brief, living cells were gated for single cells only (side scatter and forward scatter), and the samples were stained for cell populations using specific antibodies for microglia/macrophages (CD11b PeCy7; Invitrogen), oligodendrocytes (O4 APC; Miltenyi), endothelial cells (CD31 PEVio770; Miltenyi), thrombocytes/immune cells (CD45 FITC; Biolegend), and astrocytes (astrocyte cell surface antigen-2 [ACSA-2] PE, Miltenyi; Table 1). Neuronal purity of 90–96% was achieved.

Hanuscheck N., Thalman C., Domingues M., Schmaul S., Muthuraman M., Hetsch F., Ecker M., Endle H., Oshaghi M., Martino G., Kuhlmann T., Bozek K., van Beers T., Bittner S., von Engelhardt J., Vogt J., Vogelaar C.F, & Zipp F. (2022). Interleukin-4 receptor signaling modulates neuronal network activity. The Journal of Experimental Medicine, 219(6), e20211887.

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Quantifying Infiltrating Leukocytes in HSV-1 Infection

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Mice were inoculated with 3 x 10⁴ PFU HSV-1 KOS or mock-infected and sacrificed by transcardial perfusion with PBS at day 3 post-infection. Brains were dissociated using the Neural Tissue Dissocation Kit (P) (Miltenyi Biotec). For enumeration of infiltrating leukocytes, cells were first treated with TruStain FcX anti-CD16/32 antibody (Fc block-BioLegend; 93) before staining with the following antibodies: Ly6G APC-Cy7 (BioLegend; RB6-8C5), CD11c APC (BioLegend; N418), and CD11b BV421 (BD; M1/70). Flow cytometry was performed on a FACS CantoII (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo 10.1 software (Ashland, OR, USA).
For fluorescently-labeled inhibitor of caspase activity (FLICA) assays, cell suspensions were first incubated with a 1:1000 dilution of Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) before incubation with the FAM-FLICA reagent according to the manufacturer’s protocol (ImmunoChemistry Technologies). Cells were treated with an Fc-blocking reagent (Fc block-BioLegend; 93) and stained with the following antibodies before being analyzed: ACSA-2 PE (Miltenyi Biotec; REA969), CD45 PerCP-Cy 5.5 (BD Biosciences; 30-F11), and CD11b PE-Cy7 (eBioscience; M1/70). Gating of positive populations was based on Fluorescence minus one control (FMO) samples.

Hayes C.K., Wilcox D.R., Yang Y., Coleman G.K., Brown M.A, & Longnecker R. (2021). ASC-dependent inflammasomes contribute to immunopathology and mortality in herpes simplex encephalitis. PLoS Pathogens, 17(2), e1009285.

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