Purified LPS (E. Coli 0111:B4, catalog no. L4391) was obtained from Sigma-Aldrich (St. Louis, MO). Recombinant IFNα-2b (Intron® A) was obtained from Schering Corp. (Bloomfield, N. J.) Allophycocyanin (APC)-conjugated anti-human CCR7, PerCP-eFluor 710 anti-CD11c and appropriate isotype controls were obtained from e-Bioscience (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD2, phyco-erythrin (PE) anti-CD16, anti-HLA-DR V450, Alexa fluor 700 anti-CD56, lineage cocktail 1, 7-Aminoactinomycin D (7-AAD) and appropriate isotype controls were purchased from BD Biosciences (San Jose, CA). Pacific Orange anti-CD3, and Quantum dots 800 anti-CD14 and corresponding isotype controls were purchased from Invitrogen Corp. (San Diego, CA). ECD IOT Test anti-CD16, and ECD IOT Test anti-CD19, and isotype controls were purchased from Beckman Coulter Inc. (Fullerton, CA). PerCP-Cy5.5 anti-blood dendritic cell antigen (BDCA)-1 (or CD1c), FITC anti-human DC-SIGN, and corresponding controls were obtained from BioLegend (San Diego, CA). FITC anti-BDCA-2 (CD303), PE anti-BDCA-3, PE-anti-BDCA-4, corresponding controls and Fc Receptor Blocking Reagent human were purchased from Miltenyi Biotec (Auburn, CA).
Lineage cocktail 1
Manufactured by BD
Sourced in United States
Lineage cocktail 1 is a laboratory reagent used for cell culture applications. It is designed to maintain the undifferentiated state of stem cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
7 aminoactinomycin d 7 aad, by BD (1 mentions)
Anti hla dr v450, by BD (1 mentions)
Cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Cd8 apc, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cd95 pe, by BD (1 mentions)
Facscanto, by BD (1 mentions)
Hla dr, by BD (1 mentions)
Bdca2, by Miltenyi Biotec (1 mentions)
3 protocols using lineage cocktail 1
1
Immunophenotyping of Innate Immune Cells
2
Apoptosis Analysis of Activated T Cells and DCs
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The cells were harvested on day 5 of MLR, and apoptosis was determined by staining cells with a combination of Annexin-V FITC with propidium iodide using the Annexin-V FITC Apoptosis Detection Kit (BD Pharmingen, USA), according to the manufacturers’ protocols. The cells were co-stained with CD3-APC. Additionally, cells were labelled with CD4-FITC (clone: RPA-T4, eBioscience, USA), CD8-APC (clone: RPA-T8, eBioscience, USA), CD95-PE (clone: DX2, BD Pharmingen, USA) or CD95L-PE (clone: NOK-1, BioLegend, USA) to identify T cells and CD11c-APC, CD95L-PE, Lineage Cocktail 1 (lin 1; CD3, CD14, CD16, CD19, CD20, CD56)-FITC (BD Pharmingen, USA) to identify DCs. The results were acquired and analyzed as described above.
3
Phenotypic Characterization of Plasmacytoid Dendritic Cells
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To evaluate the percentage of pDCs, PBMCs (3 • 10 5 ) were stained with the Lineage cocktail 1 (BD Pharmingen) containing Abs that, in combination, stain lymphocytes, monocytes, eosinophils, and neutrophils. In the Lineage negative fraction of a lympho-mono gate, we considered as pDCs the cells triple positive for IL3Ra (CD123), HLA-DR (BD Pharmingen), and BDCA2 (Miltenyi Biotec). The full gating strategy is shown in Supplementary Fig. S1 (Supplementary Data are available online at www.liebertpub .com/jir).
The expression on the pDC surface of CD86 (BD Pharmingen) or inducible T cell co-stimulator ligand (ICOS-L), B7-H1, Ig-like transcript 7 (ILT7), Fce-RIg (CD23) and Fcg-RIIa (CD32) (eBioscience) was evaluated in the Lin -CD123 + HLA-DR + BDCA2 + gate by running cells on a FACSCanto (BD Pharmingen) and analyzing data by FlowJo software (TreeStar, Inc.) .
Monoclonal Abs and IgG1, IgG2a control Abs (BD Pharmingen) were used conjugated with FITC, PE, PERcP, APC, or APC-Cy7 as needed.
The expression on the pDC surface of CD86 (BD Pharmingen) or inducible T cell co-stimulator ligand (ICOS-L), B7-H1, Ig-like transcript 7 (ILT7), Fce-RIg (CD23) and Fcg-RIIa (CD32) (eBioscience) was evaluated in the Lin -CD123 + HLA-DR + BDCA2 + gate by running cells on a FACSCanto (BD Pharmingen) and analyzing data by FlowJo software (TreeStar, Inc.) .
Monoclonal Abs and IgG1, IgG2a control Abs (BD Pharmingen) were used conjugated with FITC, PE, PERcP, APC, or APC-Cy7 as needed.
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