Aria plus high speed sorter

Manufactured by BD

Sourced in United States

The BD-Aria-Plus is a high-speed cell sorter designed for use in flow cytometry applications. It is capable of sorting cells at high rates while maintaining high purity and recovery. The core function of the BD-Aria-Plus is to detect and physically separate individual cells based on their measured characteristics.

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Lab products found in correlation

Apc conjugated anti mouse ly6g antibody, by BD (2 mentions) Cd11b apc cy7, by BD (2 mentions) Anti mouse ly6g pe, by BD (2 mentions) Apc cy7 anti mouse cd11b, by BD (2 mentions) Calcium ionophore, by Merck Group (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions) Ab20669, by Abcam (1 mentions) Apc conjugated anti cd11b monoclonal antibody, by BD (1 mentions) Alexa fluor 555 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)

5 protocols using aria plus high speed sorter

Isolation of Mouse Neutrophils

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Mouse neutrophils were isolated from bone marrow of tibias and femurs (18 ). Neutrophils were sorted on a BD-Aria-Plus high-speed sorter after incubation with APC-conjugated anti-mouse-Ly6G antibody (BD Biosciences) and APC-Cy7 CD11b (BD Biosciences) (purity >96% and >95% viability by Tryptan blue staining) (Supplementary Fig. 1).

Tohme S., Yazdani H.O., Al-Khafaji A.B., Chidi A.P., Loughran P., Mowen K., Wang Y., Simmons R.L., Huang H, & Tsung A. (2016). Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress. Cancer research, 76(6), 1367-1380.

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Murine Neutrophil Isolation and NET Generation

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Murine neutrophils were isolated from bone marrow as previously described [24 (link)]. A BD Aria Plus high-speed sorter was used to sort neutrophils after incubation with PE-anti-mouse-Ly6G and APC-Cy7-anti-mouse-CD11b (BD Bioscience, Franklin Lakes, NJ, USA). To generate NETs, neutrophils were plated to adhere to coated plates for 1 h before 4 h stimulation with Calcium Ionophore (A23187, 5 µM; Sigma-Aldrich, St. Louis, MO). Neutrophils re-suspended in RPMI were also stimulated as described above for NET formation in cell culture dishes. After discarding the supernatant, NETs were harvested in 5 mL of new medium and centrifuged at 300× g for 10 min to pellet intact cells. Then, the supernatant was further centrifuged at 20,000× g for 30 min to pellet NETs. Washed NETs were then resuspended in 1 mL of RPMI 1640 with 1% BSA. Nucleosomes and cell-free DNA were measured in washed NET preparations to confirm the presence of NETs [24 (link)].

Zhang H., Wang Y., Onuma A., He J., Wang H., Xia Y., Lal R., Cheng X., Kasumova G., Hu Z., Deng M., Beane J.D., Kim A.C., Huang H, & Tsung A. (2021). Neutrophils Extracellular Traps Inhibition Improves PD-1 Blockade Immunotherapy in Colorectal Cancer. Cancers, 13(21), 5333.

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Neutrophil Isolation from Murine Tissues

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Neutrophils were isolated from peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) of APC-WT and APC-KRAS^G12D mice. Neutrophils were sorted on a BD-Aria-Plus high-speed sorter after incubation with APC-conjugated anti-mouseLy6G antibody (BD Biosciences, San Jose, California, USA) and APC-Cy7 CD11b (BD Biosciences, San Jose, California, USA).

Shang A., Gu C., Zhou C., Yang Y., Chen C., Zeng B., Wu J., Lu W., Wang W., Sun Z, & Li D. (2020). Exosomal KRAS mutation promotes the formation of tumor-associated neutrophil extracellular traps and causes deterioration of colorectal cancer by inducing IL-8 expression. Cell Communication and Signaling : CCS, 18, 52.

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Isolation and NET Formation from Murine Neutrophils

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Murine neutrophils were isolated from bone marrow of tibias and femurs as previously described (18 (link), 28 (link)). A BD Aria Plus high-speed sorter was used to sort neutrophils after incubation with PE-anti-mouse-Ly6G and APC-Cy7-anti-mouse-CD11b (BD Bioscience). To generate NETs, neutrophils were then plated to adhere in coated plates for 1 h before 4 h stimulation with phorbol 12-myristate 13-acetate (PMA, 30 nM; Sigma-Aldrich). Neutrophils re-suspended in RPMI were also stimulated as described above for NETs formation in cell culture dishes. After discarding the supernatant, NETs were harvested in 5 ml of new medium and centrifuged at 300 g for 10 min to pellet intact cells. Then, the supernatant was further centrifuged at 20,000 g for 30 min to pellet NETs. Washed NETs were then resuspended in 1 ml of RPMI 1640 + 1% BSA. Nucleosomes and cell-free DNA were measured in washed NET preparations to confirm the presence of NETs (29 (link)).

Zhang H., Goswami J., Varley P., van der Windt D.J., Ren J., Loughran P., Yazdani H., Neal M.D., Simmons R.L., Zhang J., Tsung A, & Huang H. (2020). Hepatic Surgical Stress Promotes Systemic Immunothrombosis That Results in Distant Organ Injury. Frontiers in Immunology, 11, 987.

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Neutrophil Isolation and NET Formation

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The bone marrow of the femur and tibia were collected from WT mice as previously described by Cools-Lartigue et al (15 ). Neutrophils were sorted with a BD-Aria-Plus high-speed sorter using APC conjugated anti-CD11b monoclonal antibody and APC-Cy7 conjugated anti-Lys6G monoclonal antibody (both BD Pharmingen). 2×10⁶ neutrophils were plated onto gelatin coated 6cm dishes and onto gelatin coated glass cover slips, and allowed to attach to the plate during a 1 hour incubation period at 37°C. The FFA palmitic (C16:0), linoleic (C18:2), and oleic acid (C18:1) (all from Nu-Check Prep) were dissolved in 25% FFA-free BSA and were added in a final concentration of 50uM to stimulate the neutrophils. LPS at 100nM and H₂O₂ (both Sigma) were used as positive controls, whereas 25% BSA without FFA served as negative control. After 4 hours of stimulation at 37°C, the supernatants were spun and frozen for analysis of MPO-DNA levels. Cover slips were stained with antibodies against histone 2AX (Abcam ab20669, with Alexa Fluor 555 goat anti-rabbit secondary antibody from Life Technologies), actin (Alexa Fluor 488, Invitrogen) and DAPI, and NET formation was visualized with an Olympus Fluoview 1000 microscopic camera.

van der Windt D.J., Sud V., Zhang H., Varley P.R., Goswami J., Yazdani H.O., Tohme S., Loughran P., O’Doherty R.M., Minervini M.I., Huang H., Simmons R.L, & Tsung A. (2018). NEUTROPHIL EXTRACELLULAR TRAPS PROMOTE INFLAMMATION AND DEVELOPMENT OF HEPATOCELLULAR CARCINOMA IN NON-ALCOHOLIC STEATOHEPATITIS. Hepatology (Baltimore, Md.), 68(4), 1347-1360.

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