HTATIP2 protein expression of isolated Mo/siRNA-treated TIE2-iBMMs was analyzed by flow cytometry. For human proarteriogenic Mo/MΦs, following FcR blocking (Miltenyi Biotec), cells were stained for surface expression of CD14 (BD Biosciences) and TIE2 (R&D Systems). Washed cells were subsequently fixed in 2% paraformaldehyde for 15 minutes at room temperature, permeabilized with Perm Buffer IV for 20 minutes (BD Biosciences), and stained for intracellular HTATIP2 (Abcam). Secondary antibody staining was then conducted using an APC-conjugated secondary antibody (Jackson ImmunoResearch) (Supplemental Table 10 ). Cells were subsequently washed, resuspended in buffer (PBS, 0.5% BSA, 2 mM EDTA), and analyzed using a MACSQuant flow cytometer (Miltenyi Biotec). Intracellular protein expression was quantified in terms of MFI using FlowJo V10 software.
Apc conjugated secondary antibody
Manufactured by Jackson ImmunoResearch
APC-conjugated secondary antibody is a laboratory reagent used in various immunoassays and flow cytometry applications. It is designed to detect and visualize primary antibodies bound to target analytes. The APC (allophycocyanin) fluorescent dye is conjugated to the secondary antibody, allowing for sensitive detection and quantification of the target of interest.
Automatically generated - may contain errors
Lab products found in correlation
Fcr blocking, by Miltenyi Biotec (1 mentions)
Tie 2, by R&D Systems (1 mentions)
Macsquant flow cytometer, by Miltenyi Biotec (1 mentions)
Perm buffer 4, by BD (1 mentions)
Hoechst 33342, by Bio-Rad (1 mentions)
Grp78 antibody, by Santa Cruz Biotechnology (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Anti cd45 percp, by Exbio (1 mentions)
Annexin 5 fitc, by Exbio (1 mentions)
Lsrfortessa analyzer, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
3 protocols using apc conjugated secondary antibody
1
Flow Cytometric Analysis of HTATIP2 Expression
2
Confocal Microscopy of Intracellular and Extracellular Protein Interactions
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Confocal microscopy was performed according to the LSM 780 user manual (Zeiss, Oberkochen, Germany) and the μ-slide 8 well user instruction (ibidi®, Martinsried, Germany). Briefly, parental OECM1 and FaDu cells were seeded in 8-well μ-slides one day before immunofluorescence staining was performed. The nuclei were stained with Hoechst 33342 (Bio-Rad). For the interactome candidates that are at the intracellular side of the plasma membrane, the live cells were incubated with the GRP78 antibody (Santa Cruz) and the APC conjugated secondary antibody (JacksonImmunoResearch), followed by fixation with 1% formaldehyde. Cell were then permeabilized with 0.5% Tween 20, followed by incubation with primary antibodies recognizing the interactome candidates and the appropriate PerCP-conjugated secondary antibody (JacksonImmunoResearch). For extracellular interactome candidates, live cells were not fixed nor permeabilized before or after antibody staining. PBS containing 5% FBS was added to the cells, and then the fluorescence images were acquired using an LSM780 confocal microscope (Zeiss).
3
Characterization of Immune Subsets in AML
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Peripheral blood mononuclear cell (PBMCs) isolated from AML patients or C57BL/6 (B6) mice, as well as mouse splenocytes, bone-marrow derived DCs and tumor cells were stained with panels of fluorescent antibodies to evaluate the abundance, phenotype and function of immune cell subsets (Online Supplementary Table S1-2). Briefly, cells were incubated with primary antibodies or appropriate isotype controls for 20 min at 4 °C. For the analysis of CRT levels on AML blasts, PBMCs were labeled with anti-CD45 PerCP (Exbio) and anti-CD33 PE monoclonal antibodies (BioLegend). Malignant blasts from AML patients were defined as CD45+ cells expressing high levels of CD33 (CD33high). Surface CRT staining was performed by a three-step procedure: (1) incubation with primary CRT-specific antibody (Enzo Life Sciences), (2) incubation with an APC-conjugated secondary antibody (Jackson Immunoresearch Laboratories) and (3) incubation with Annexin V-FITC (Exbio) and 4′,6-diamidino-2-phenylindole (DAPI, from Molecular Probes) to assess the cell viability. Surface-exposed CRT levels were analyzed only on live (AnnV−DAPI−) and dying (AnnV+/DAPI−) but not dead (DAPI+) cells. Flow cytometry data were acquired on the LSRFortessa analyzer (BD Biosciences) and analyzed with the FlowJo software package (Tree Star, Inc.).
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