Facs aria iitm

Manufactured by BD

The BD FACS Aria-IITM is a high-performance cell sorter designed for advanced flow cytometry applications. It features a multi-laser optical system and high-speed cell sorting capabilities for precise analysis and sorting of complex cell populations.

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Lab products found in correlation

Rosettesep human monocyte enrichment kits, by STEMCELL (2 mentions) Cd4 cd127low t cell enrichment kits, by STEMCELL (2 mentions) Anti cd14 conjugated microbeads, by Miltenyi Biotec (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Collagenase, by Thermo Fisher Scientific (1 mentions) Ultra fine, by BD (1 mentions) Pan dendritic cell isolation kit, by Miltenyi Biotec (1 mentions) αcd3 okt3, by BioLegend (1 mentions)

6 protocols using facs aria iitm

Tissue Macrophage Isolation from Infected Mice

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The mice were injected intradermally in the ear pinna either with PBS (uninfected naïve mice) or 10⁶ stationary phase LdWT/ LdCen^−/− promastigotes. In each study, a minimum of six mice were used per group. Age-matched naive mice were used as a control. At 6h post infection, mice were sacrificed and tissue resident macrophages (Cd11b⁺F4/80⁺ Ly6G⁻ Ly6C⁻ MHCII⁻) from different groups of mice were sort selected using the BD FACS Aria-IITM. Single-cell suspensions were prepared from ear dermis, and then labeled for fluorochrome tagged anti–TCR-β, anti-NK1.1, anti-Cd19 Abs using fluorochrome tagged magnetic beads, and passed through LS columns to select out these cell types. Flow through enriched population was collected and stained with Cd11b-BV510, F4/80-PAC, Ly6G-APC, Ly6C- FITC, MHCII- APC-CY7 Ab and further sort selected.

Bhattacharya P., Dey R., Saxena A., Karmakar S., Ismail N., Gannavaram S., Dagur P.K., Satoskar M., Satoskar S., De Paoli S., Takeda K., McCoy JP J.r, & Nakhasi H.L. (2020). Essential role of neutrophils in the protective immune response induced by a live attenuated Leishmania vaccine. Journal of immunology (Baltimore, Md. : 1950), 205(12), 3333-3347.

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Leishmania Infection Immune Response

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The mice were injected intradermally in the ear pinna either with PBS (uninfected naïve mice) or 10⁶ stationary phase LdWT/ LdCen^−/− promastigotes. In some experiment’s mice were infected with LdWT^RFP/ LdCen^{−/−mCherry} parasites. In each study, a minimum of six mice were used per group. At different time post infection mice were sacrificed and total neutrophils (Cd11b⁺ Ly6G⁺ Ly6C^int) or parasitized neutrophils (Cd11b⁺ Ly6G⁺ Ly6C^intRFP⁺/mCherry⁺), macrophages (Cd11b⁺Ly6C⁻Ly6G⁻CD11c⁻MHCII⁺) and DCs (Cd11b⁺Ly6C⁻Ly6G⁻CD11c⁺MHCII⁺) from different groups of mice were sort selected using the BD FACS Aria-IITM. Single-cell suspensions were prepared from ear dermis, ear dLN and spleen then labeled for fluorochrome tagged anti–TCR-β, anti-NK1.1, anti-Cd19 Abs using fluorochrome tagged magnetic beads, and passed through LS columns to select out these cell types. Flow through enriched population was collected and stained with neutrophil, macrophages and dendritic cell specific markers and further sort selected.

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Isolation of Immune Cells from Murine Leishmaniasis Model

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Using a 29-gauge needle (BD Ultra-Fine) and a volume of 10 μL, 3 × 10⁶ LdWT/LdCen^−/− parasites were intradermally (ID) injected into C57BL/6 mice in the ear pinna. Each study employed a minimum of six mice per group. As a control group, naïve mice that were age-matched received PBS. Macrophages (MØ) (Cd11b⁺Ly6G⁻Ly6c⁻Cd11c⁻MHCII⁺) and dendritic cells (DC) (Cd11b⁺Ly6G⁻Ly6c⁻Cd11c⁺MHCII^hi) were sort selected from the spleen and ear dLN of LdWT and LdCen^−/− infected mice at 72 h and 7 days after infection, respectively, by high-speed FACS cell sorter system (BD FACS ARIA IITM). Briefly, tweezers and a syringe plunger were used to physically separate and remove the retro maxillary (ear-draining) lymph nodes. Filtration of tissue homogenates using a 70-μm cell strainer was performed (Falcon Products, Corning, NY, USA). To create a single-cell suspension, mouse spleens were collected and processed with collagenase (1 mg/mL) and DNase I (20 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA). The single cell suspension from the spleen and dLN of the ear was labeled with anti-TCR-, anti-NK1.1, and anti-Cd1b that had been APC-tagged. To exclude certain cell types, anti-APC magnetic beads were utilized and run across LS columns. Flow through enriched population of macrophages and DCs were collected and stained with macrophages and DC-specific markers and then further sorted.

Bhattacharya P., Gannavaram S., Ismail N., Saxena A., Dagur P.K., Akue A., KuKuruga M, & Nakhasi H.L. (2023). Toll-like Receptor-9 (TLR-9) Signaling Is Crucial for Inducing Protective Immunity following Immunization with Genetically Modified Live Attenuated Leishmania Parasites. Pathogens, 12(4), 534.

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Splenic DC Infection and Parasite Load in Aged Mice

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The young and aged mice were infected via tail vein with 3 X 10⁶ stationary phase red fluorescent LdWT RFP, or LdCen-/-m-cherry promastigotes. In each study, at least 6 mice were used per group. Age-matched naive mice used as control. At day 4 post infection, mice were sacrificed and parasite load was recorded from spleens of the LdWT and LdCen-/- infected mice by culturing the separated host cell preparations by limiting dilutions as previously described [40 (link)].
In a separate experiment the splenic DCs were sort selected. Single-cell suspensions were prepared from spleens, and RBCs were lysed using ACK lysing buffer. Dendritic cells were enriched using pan dendritic cell isolation kit (Miltenyi Biotec) and subsequent sort selecting of Leishmania (RFP/m-Cherry) infected splenic DCs and uninfected splenic DCs from different age groups of infected mice were done by high speed FACS cell sorter system (BD FACS Aria-IITM). Infected splenic DCs were sorted by gating live single enriched DCs for Cd11c+Ly6G-Cd11b-RFP/m-Cherry⁺. Uninfected splenic DCs after enrichment [Cd11c+ Ly6G-Cd11b- RFP/ m-Cherry^-] were also sort selected.

Bhattacharya P., Dey R., Dagur P.K., Joshi A.B., Ismail N., Gannavaram S., Debrabant A., Akue A.D., KuKuruga M.A., Selvapandiyan A., McCoy JP J.r, & Nakhasi H.L. (2016). Live Attenuated Leishmania donovani Centrin Knock Out Parasites Generate Non-inferior Protective Immune Response in Aged Mice against Visceral Leishmaniasis. PLoS Neglected Tropical Diseases, 10(8), e0004963.

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Isolation and Purification of Human Immune Cells

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PBMCs were isolated from buffy coats/LRS chambers obtained from healthy adult blood donors at the Stanford Blood Center. CD14⁺ monocytes and CD4⁺CD127^low T cells were isolated from PBMCs using RosetteSep Human Monocyte Enrichment Kits and CD4⁺CD127^low T cell Enrichment Kits, respectively (Stem Cell Technologies). CD14⁺ monocytes were further purified to > 97% purity by magnetic separation with anti-CD14 conjugated microbeads (Miltenyi Biotec). Memory CD4⁺ Th and Tregs were obtained by sorting pre-enriched T cells with fluorescently labeled mAbs against CD4, CD25, CD127, CD45RA, CD45RO and lineage markers along with propidium iodide (PI; Life Technologies). Th were defined as PI^–Lin^–CD4⁺CD45RA^–CD45RO⁺CD127⁺CD25^–/low, and Tregs were defined as PI^–Lin^–CD4⁺CD127^low CD25⁺. Cells were sorted with a BD FACSAria II^TM after they had been stained with mAbs to CD25 (BC96), CD14 (HCD14), CD19 (H1B19), CD20 (2H7) and CD56 (HCD56) (BioLegend) and CD4 (S3.5), CD45RA (MEM-56), CD45RO (UCHL1) (Life Technologies).

Zhang X., Zheng P., Prestwood T.R., Zhang H., Carmi Y., Tolentino L.L., Wu N., Choi O., Winer D.A., Strober S., Kang E.S., Alonso M.N, & Engleman E.G. (2020). Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells. Frontiers in Immunology, 11, 1982.

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Isolation and Culture of DCreg from Monocytes

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CD14+monocytes and CD4+CD127low T-cells were isolated from PBMCs of healthy blood donors using RosetteSep^™ Human Monocyte Enrichment Kits and CD4+CD127low T-cell Enrichment Kits, respectively (Stem Cell Technologies). CD14+monocytes were further purified to >97% purity by magnetic separation with anti-CD14 conjugated microbeads (Miltenyi Biotec). Memory CD4+ Th (T helper) and Tregs were obtained by sorting pre-enriched T-cells with fluorescently labeled mAbs against CD4, CD25, CD127, CD45RA, CD45RO and lineage markers along with propidium iodide (PI; Life Technologies). Th were defined as PI-Lin-CD4+CD45RA-CD45RO+CD127+CD25-/low, and Tregs were defined as PI-Lin-CD4+CD127low CD25+. Cells were sorted with a BD FACSAria IITM after staining, as above. Monocytes (1×10⁶) were cultured with allogeneic Th and Tregs at a 10:1:1 in 12-well plates with 50ng/ml αCD3 (OKT3, BioLegend). HLA-DR+CD2- DCreg were sorted out by flow cytometry on day 4 of culture. DCreg were stimulated with 1μg/ml LPS. After 18 hr, cells were washed 3x in PBS and HLA-DR+CD2- DC were purified by FACS and incubated with allogeneic naïve CD4+ T-cells (10⁵/well) at a 1:2 DCs to T-cell ratio in the presence or absence of 2μg/ml anti-TGFβ. After 6 days, responder CD4+ T-cells were analyzed for CD25 and Foxp3 expression.

Marshall P.L., Nagy N., Kaber G., Barlow G.L., Ramesh A., Xie B.J., Linde M.H., Haddock N.L., Lester C.A., Tran Q.L., de Vries C., Hargil A., Malkovskiy A., Gurevich I., Martinez H.A., Kuipers H.F., Yadava K., Zhang X., Evanko S.P., Gebe J.A., Wang X., Vernon R.B., de la Motte C., Wight T.N., Engleman E.G., Krams S.M., Meyer E, & Bollyky P.L. (2020). Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection. Matrix biology : journal of the International Society for Matrix Biology, 96, 69-86.

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