Renal-infiltrated immune cells were screened for various cell surface and intracellular markers with flow cytometry. Briefly, 1 × 106 cells were incubated with anti-mouse CD45, F4/80, CD4, CD8, CD11c, CD11b, Ly6G, and monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Immune cells derived from the kidneys were concomitantly stained for the intracellular content of TNF-α, IL-10, IL-17, and forkhead box P3 (FoxP3) by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC) (BD Bioscience). For intracellular cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometric analysis was conducted on a BD Biosciences FACSCalibur and analyzed by using the Flowing Software analysis program.
Allophycocyanin apc
Manufactured by BD
Sourced in United States
Allophycocyanin (APC) is a fluorescent phycobiliprotein found in cyanobacteria and red algae. It functions as a light-harvesting pigment in the photosynthetic process. APC absorbs light in the orange-red region of the visible spectrum and emits fluorescence in the near-infrared region, making it a useful fluorescent label for various biological applications.
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Lab products found in correlation
Facscalibur, by BD (7 mentions)
Golgistop, by BD (4 mentions)
Phycoerythrin pe, by BD (3 mentions)
Fluorescein isothiocyanate (fitc), by BD (3 mentions)
Peridinin chlorophyll protein, by BD (2 mentions)
Anti mouse monoclonal antibodies, by BD (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Protein g magnetic beads, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Cytofix cytoperm fixation permeabilization kit, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Anti cd4 rpa t4, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
Peridinin chlorophyll protein percp, by BD (1 mentions)
C raf, by BD (1 mentions)
Cd11b, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
13 protocols using allophycocyanin apc
1
Renal Immune Cell Characterization
2
Immunophenotyping of Lung-Infiltrated Cells
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Lung-infiltrated immune cells were screened for various cell surface and intracellular markers with flow cytometry. Briefly, 1 × 106 cells were incubated with anti-mouse CD45, F4/80, CD4, CD8, CD11c, CD11b, CD49b, FasL, CD107, perforin, NKG2D monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Immune cells derived from the lungs were concomitantly stained for the intracellular content of TNF-α, IL-10, and IL-17 by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC) (BD Biosciences). For intracellular cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometric analysis was conducted on a BD Biosciences' FACSCalibur and analyzed by using the flowing software analysis program.
3
Profiling Tumor-Infiltrating Immune Cells
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Tumor-infiltrating leucocytes were investigated for different cell surface and intracellular markers with flow cytometry. Briefly, 1 × 106 cells were incubated with anti-mouse F4/80, CD4, CD8, CD11c, NK1.1, CD80, I-A, granzyme B, and Fas ligand (FasL) monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. Immune cells derived from the tumors were concomitantly stained for the intracellular content of TNF-α, IFN-γ, IL-12, IL-4, and IL-17 by using the fixation/permeabilization kit and anti-mouse monoclonal antibodies conjugated with FITC, PE, PerCP, and APC (BD Biosciences). For intracellular cytokine staining, cells were stimulated with 50 ng/mL PMA and 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) was added. Cells were fixed in Cytofix/Cytoperm, permeated with 0.1% saponin, and stained with fluorescent Abs. Flow cytometric analysis was conducted on a BD Biosciences' FACSCalibur and analyzed by using the Flowing Software analysis program [17 (link)].
4
Quantifying CD19+ B cells in lupus mice
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Peripheral blood was obtained from the hCD20-DTA/MRL/lpr and JhD/MRL/lpr mice. MRL/lpr mice were used as positive controls. Blood samples were first lysed in red blood cell (RBC) lysis buffer and washed with ice-cold 2 % FBS. B cells were stained with anti-mouse CD19 conjugated to phycoerythrin (PE) for JhD/MRL/lpr, or allophycocyanin (APC) (BD Bioscience, San Jose, CA) for hCD20-DTA/MRL/lpr (the YFP gene in the latter strain is incompatible with red fluorochromes). The percentage of CD19 positive cells was gated out of single cell lymphocyte populations by flow cytometry (FACScalibur, BD Bioscience).
5
Peripheral Blood Immunophenotyping for NK Cells
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Following the isolation of PBMCs, the cells were washed twice with PBS (Sigma-Aldrich, Germany). In the sample gating, SSC-H and FSC-H were comparatively employed to gate all lymphocytes through forward and side scatter. They were analyzed even further for their CD4+, CD8+, CD56+, and CD16+ expression.
This study conducted peripheral blood immunophenotyping assay in accordance with flow cytometry to identify Natural Killer (NK) cells. For PBMC staining, triple-color immunofluorescence analyses of the lymphocyte markers were performed through the use of anti-CD3, anti-CD16, and anti-CD56 antibodies labelled by fluorescein isothiocyanate (FITC) (BD Biosciences), phycoerythrin (PE) (BD Biosciences), and allophycocyanin (APC) (BD Biosciences) fluorochromes, respectively. The NK population comprised CD3− CD56+ CD16+ cells in the peripheral blood. Anti-CD4 and anti-CD8 fluorescent conjugated antibodies were used for evaluating CD4+ and CD8+ T lymphocytes after lymphocytes gating, respectively. The isotype controls included FITC, PE, and APC mouse IgG2a. Flow cytometry was performed using FACSCalibur (BD Biosciences, San Jose, CA, USA), and FlowJo software was then used for data analysis.
This study conducted peripheral blood immunophenotyping assay in accordance with flow cytometry to identify Natural Killer (NK) cells. For PBMC staining, triple-color immunofluorescence analyses of the lymphocyte markers were performed through the use of anti-CD3, anti-CD16, and anti-CD56 antibodies labelled by fluorescein isothiocyanate (FITC) (BD Biosciences), phycoerythrin (PE) (BD Biosciences), and allophycocyanin (APC) (BD Biosciences) fluorochromes, respectively. The NK population comprised CD3− CD56+ CD16+ cells in the peripheral blood. Anti-CD4 and anti-CD8 fluorescent conjugated antibodies were used for evaluating CD4+ and CD8+ T lymphocytes after lymphocytes gating, respectively. The isotype controls included FITC, PE, and APC mouse IgG2a. Flow cytometry was performed using FACSCalibur (BD Biosciences, San Jose, CA, USA), and FlowJo software was then used for data analysis.
6
Tumor-Infiltrating Leukocyte Profiling
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Tumor-infiltrating leukocytes were isolated form breast tumors and investigated for different cell surface and intracellular markers with flow cytometry [12 (link)]. For that purpose, 1 × 106 isolated tumor-infiltrating leukocytes were incubated with anti-mouse F4/80, CD11c, NK1.1, CD80, CD86, I-A, granzyme B, CD178, CD4, and CD8 monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (BD Biosciences, San Jose, CA, USA). For intracellular cytokine staining, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 500 ng/mL ionomycin for 5 h, and GolgiStop (BD Biosciences) and then incubated in a BD fixation/permeabilization solution (BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit) and washed in 1× BD Perm/Wash™ buffer. Fixed/permeabilized cells were stained for TNF-α, IFN-γ, IL-12, IL-4, IL-17, IL-10, and FoxP3 by using appropriate anti-mouse monoclonal antibodies conjugated with FITC, PE, PerCP, and APC (BD Biosciences, San Jose, CA, USA). BD Biosciences' FACSCalibur and Flowing Software were used for flow cytometry analysis [12 (link)].
7
PBMC and CSF Cell Activation Analysis
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Freshly isolated PBMCs and CSF cells were activated for six hours with PMA (50 ng/mL, Sigma-Aldrich), ionomycin (500 ng/mL, Sigma-Aldrich), and Golgiplug (BD Biosciences, Paris, France) before flow cytometric analysis. Cells were immunostained by surface antibody anti-CD19 (HIB19) and anti-CD4 (RPA-T4) conjugated respectively with Fluorescein isothiocyanate (FITC) and Allophycocyanin (APC) (BD Biosciences, Paris, France). Cells were fixed and permeabilized according to Cytofix/Cytoperm™ Kit protocol (BD Biosciences, Paris, France). Intracellular Phycoerythrin (PE) conjugated anti-IL-10 antibody (JES3-9D7) was added to cell suspension for 1 h in dark at +4 °C (BD Biosciences, Paris, France). Data were analyzed using the FlowJo software (version 7.6).
8
Multicolor Flow Cytometry for Immune Cell Analysis
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Flow cytometric analysis was performed using a FACSCalibur analyzer and CellQuest software (BDIS, San Jose, CA) [28 (link)]. In summary for each sample, 50 μl of blood was stained with BD monoclonal antibodies conjugated with immunofluorescence such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE), or Peridinin Chlorophyll Protein Complex (PerCP) or Allophycocyanin (APC), (BD Biosciences, San Jose, CA) using lyse no wash sample preparation method, with lymphocyte gating done according to the manufacturer’s instructions. CD3-FITC, CD8-PE, CD45-PerCP, and CD4-APC were used to label T cells in the first sample. In the second sample, CD3-FITC, CD56/16-PerCP, and CD19-APC monoclonal antibodies were used for identification of natural killer (NK) cells and B cells. The percentages of CD3+/CD4+ (helper T-cell), CD3+/CD8+ (cytotoxic T-cell), CD3−/CD56/16+ (natural killer cell), and CD3−/CD19+ (B-cell) cells were determined by flow cytometric analysis.
9
Antibody Characterization for Flow Cytometry
10
Flow Cytometric Analysis of Immune Cells
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Antibodies for flow cytometric analysis, PE-conjugated CD38 (clone HIT2) and APC-conjugated CD11b (clone ICRF44) conjugated with allophycocyanin (APC), were from BD Biosciences (San Jose, CA, USA). SLP-76, Lyn, Fgr, Vav1, p-tyr, HRP anti-mouse and anti-rabbit antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-c-Cbl (clone C-15, catalogue number sc-170, lot H0414) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NUMB Antibody catalogue number (703078) was from Thermo Fisher Scientific (Waltham, MA, USA). The c-Raf antibody were from BD Biosciences (San Jose, CA, USA). Protease and phosphatase inhibitors were purchased from Sigma (St. Louis, MO, USA). Protein G magnetic beads used for immunoprecipitation were from Millipore (Billerica, MA, USA).
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