The largest database of trusted experimental protocols

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.

Automatically generated - may contain errors

6 protocols using facs aria iitm

1

Tissue Macrophage Isolation from Infected Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
2

Leishmania Infection Immune Response

Check if the same lab product or an alternative is used in the 5 most similar protocols
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 LdWTRFP/ 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+ Ly6Cint) or parasitized neutrophils (Cd11b+ Ly6G+ Ly6CintRFP+/mCherry+), macrophages (Cd11b+Ly6CLy6GCD11cMHCII+) and DCs (Cd11b+Ly6CLy6GCD11c+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.
+ Open protocol
+ Expand
3

Isolation of Immune Cells from Murine Leishmaniasis Model

Check if the same lab product or an alternative is used in the 5 most similar protocols
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+Ly6GLy6cCd11cMHCII+) and dendritic cells (DC) (Cd11b+Ly6GLy6cCd11c+MHCIIhi) 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.
+ Open protocol
+ Expand
4

Splenic DC Infection and Parasite Load in Aged Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand
5

Isolation and Purification of Human Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
PBMCs were isolated from buffy coats/LRS chambers obtained from healthy adult blood donors at the Stanford Blood Center. CD14+ monocytes and CD4+CD127low T cells were isolated from PBMCs 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 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 PILinCD4+CD45RACD45RO+CD127+CD25–/low, and Tregs were defined as PILinCD4+CD127low CD25+. Cells were sorted with a BD FACSAria IITM 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).
+ Open protocol
+ Expand
6

Isolation and Culture of DCreg from Monocytes

Check if the same lab product or an alternative is used in the 5 most similar protocols
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.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!