Hepatic leukocytes were isolated as described previously [24 (link)]. Briefly, liver tissues were minced and sieved through a 70 μm filter. Hepatic leukocytes were purified by centrifugation on a 40% Percoll gradient (GE Healthcare), and red blood cells were lysed by RBC lysis buffer. Collected leukocytes were further enriched using PE-anti-F4/80 (BD Pharmingen, Cat# 565410) and anti-PE microbeads (Miltenyi Biotec) to isolate macrophages. Hepatocytes were isolated as described previously [25 (link)]. Briefly, the liver was perfused with a solution of EGTA and digested with a 0.075% collagenase solution. The viable hepatocytes were separated by 40% Percoll solution with centrifugation at 420 × g for 10 min at 4 °C.
Pe anti f4 80
Manufactured by BD
Sourced in United States
PE-anti-F4/80 is a fluorochrome-conjugated antibody that binds to the F4/80 antigen, which is expressed on the surface of mature tissue macrophages. This product can be used for the identification and enumeration of macrophage populations in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11b fitc, by BD (3 mentions)
Anti cd11b bv510, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd16 32, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Anti cd4 pe, by BD (2 mentions)
Anti cd11c apc, by BD (2 mentions)
Anti cd45 fitc, by BD (2 mentions)
Anti cd11b pe cy7, by BD (2 mentions)
Anti cd3 bv510, by BD (2 mentions)
Anti cd11b apc, by BD (2 mentions)
Percoll gradient, by GE Healthcare (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Liberase, by Roche (1 mentions)
Dnase 1, by Roche (1 mentions)
Fitc conjugation kit, by Abcam (1 mentions)
Anti cd3 fitc, by BD (1 mentions)
Anti tnf α fitc, by BD (1 mentions)
Fixation and permeabilization buffer, by BD (1 mentions)
Facsverse, by BD (1 mentions)
14 protocols using pe anti f4 80
1
Isolation and Purification of Hepatic Leukocytes and Hepatocytes
2
Lung Single Cell Preparation and Intracellular Staining
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Lung single cells were prepared as previously described (Longhi et al., 2009 (link)). Briefly, lungs were digested in RPMI 1640 supplemented with 20μg/L Liberase (Roche) and 25μg/L DNase I (Roche) at 37°C for 30min. After red blood cells were removed using the ammonium-chloride-potassium buffer, lung single cells were blocked using anti-CD16/32 (553142, BD Biosciences) and stained with BV510-anti-CD11b (562950, BD Biosciences), APC-anti-CD11c (17-0114-81, eBiosciences), PE-anti-F4/80 (565410, BD Biosciences), BV650-anti-Ly6G (740554, BD Biosciences), and PE-Cy7-anti-Ly6C (4341610, Invitrogen). The cells were subsequently fixed and permeablized using BD CytoFix/CytoPerm Kit (BD Biosciences) according to the manufacturer’s instructions. Intracellular virus staining was performed using an anti-IAV M2 antibody (Abcam) and a FITC conjugation kit (Abcam). Flow cytometry was performed using BD LSRFortessa (BD Biosciences) and raw data were analyzed using FlowJo software.
3
Immunophenotyping of Blood Mononuclear Cells
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Single-cell suspensions of blood samples were washed with ice-cold PBS for three times and subsequently treated with FCM lysing solution for 15 min. Then, cells were washed with ice-cold PBS to remove the excess FCM lysing solution and stained with PE-anti-F4/80, FITC-anti-CD45, FITC-anti-CD3, APC-anti-CD11c, and PE-anti-CD4 antibodies from BD Bioscience (San Diego, USA) in the dark at RT for 15 min. The data were analyzed with FlowJo software (San Carlos, CA). The portion of mononuclear macrophage (CD45+, F4/80+), T helper cells (CD3+, CD4+) and dendritic cells (CD45+, CD11c+) were determined by flow cytometry.
4
Immunophenotyping of Lung Cells
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Isolated cells in the lung were stained with indicated antibodies for further analysis. For immunostaining, the cells were washed two times with PBS containing 2% FBS, adjusted to approximately 1 × 105 to 1 × 106 cells in 100 μL of the same buffer, and labeled with PE-anti-F4/80 (BD Bioscience, 565410), PE-Cy7-anti-CD11b (BD Bioscience, 561098), and FITC-anti-TNF-α (BD Bioscience, 554418). Incubations with antibodies were performed for 30 min at 4 °C in the dark, and then cells were centrifuged for 4 min at 900 rpm. After removal of the supernatant, the cell pellet was washed once with PBS containing 2% FBS and then resuspended in fixation and permeabilization buffer (BD Bioscience, Franklin Lakes, NJ, USA) for 30 min at 4 °C. Cells were washed two times with PBS containing 2% FBS and then acquired by FACSVerse (BD Bioscience) and analyzed using software Flow Jo (Tree Star, Inc., Ashland, OR, USA).
5
Flow Cytometry Analysis of Tumor-Infiltrating Immune Cells
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For flow cytometry analysis, harvested tumors were minced into small pieces with scissors and incubated in a digestion buffer at 37 °C for 70 min. The suspensions were filtrated using a cell strainer (BD Falcon, New Jersey, USA) and centrifuged at 2000 rpm for 5 min. The collected cells were re-suspended in ACK Lysis Buffer to remove red blood cells. Next, the obtained cells were resuspended in PBS and stained by Fixable Viability Dye eFluorTM 780 (Thermo Fisher Scientific Co., Ltd., Shanghai, China) to exclude dead cells. Then, cells were washed with PBS and stained with anti-CD16/CD32 antibody (BD, New Jersey, USA) for Fc blocking to prevent nonspecific binding. Subsequently the cells were incubated with Percp-Cy5.5-anti-CD45, BV510-anti-CD3, FITC-anti-CD4, BV421-anti-CD8, PE-anti-B220, APC-anti-CD11c, FITC-anti-CD11b, PE-anti-F4/80, BV650-anti-CD86 and PE-Cy7-anti-CD206 (BD, Shanghai, China) to specifically label the T lymphocytes, B lymphocytes, DCs and Macrophage. Expect Fixable Viability Dye eFluorTM 780 and anti-CD16/32. All the above staining and centrifugation processes were carried out at 4 °C under darkness. The samples were analyzed by FCM (Sony ID7000™, Tokyo Metropolis, Japan).
6
Bronchoalveolar Lavage and Flow Cytometry
7
Multiparametric Immune Cell Analysis
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Splenocytes and lung cells were incubated with anti-CD16/32 (BD Bioscience, USA) for 30min at 4°C in PBS supplemented with 2% FBS (FACS buffer) to block non-specific binding with Fc receptors. Cells were surface-stained with BV650-anti-CD8 (BD-563234, USA), BV711-anti-CD4 (BD-563050, USA), BV510anti-CD3 (BD-563024, USA), PECY7-anti-CD25 (eBioscience-25-0251-82, USA), PE-anti-F4/80 (BD-565410, USA), BV510-anti-CD11b (BD-562950, USA), APC-anti-CD11c (eBioscience-17-0114-81, USA) for 20 min on ice and washed twice with PBS. For intracellular staining, cells were fixed and permeablized using BD CytoFix/CytoPerm Kit (BD Bioscience, USA) and stained with APC-anti-IL-4 (eBioscience-17-7041-82, USA), PE-anti-IFN-γ (eBioscience-12-7311-82, USA), PE-anti-IL-2 (eBioscience-12-7021-82, USA), BV421-anti-IL-17 (BD-563354, USA), PECY7-anti-TNF-ɑ (BD-557644, USA), Pacific blue-anti-Foxp3 (eBioscience-48-5773-82, USA) for 20min. Cells were washed with 1×BD Perm/Wash buffer and were finally resuspended in 4% paraformaldehyde (PFA) for flow cytometry analysis. Flow cytometry was performed using BD LSR Fortessa (BD Bioscience, USA) and raw data was analyzed using FlowJo software.
8
Flow Cytometry Analysis of BALF Cells
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Single-cell suspensions of BALF were obtained and blocked with antimouse CD16/32 (BD Biosciences, San Jose, CA, USA) for 10 minutes on ice and stained with antibodies against cell surface molecules or their corresponding mouse immunoglobulin G isotype for 30 minutes on ice. Anti-CD11b-APC, anti-F4/80-PE, and anti-Ly6G-fluorescein isothiocyanate (FITC) were purchased from BD Biosciences. After washing, the cells were resuspended in 400 μl PBS. Fluorescence was detected by flow cytometry (FACSCalibur, BD Biosciences).
9
Multiparametric Flow Cytometry of Splenocytes and Tumor-Infiltrating Cells
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Splenocytes were labeled with the following antibodies: anti-CD4-allophycocyanin (APC), anti-CD8-fluorescein isothiocyanate (FITC), anti-CD44-APC, anti-CD45-FITC, anti-CD49-phycoerythrin (PE), and anti-F4/80-PE and the vital dye 7-aminoactinomycin D (7-AAD) (BD Biosciences, San Jose, CA, USA). Forkhead homeobox protein 3 (Foxp3) was labeled using the anti-mouse Foxp3-FITC staining kit (eBioscience) according to the manufacturer’s instructions. For analysis of tumor-infiltrating cells, tumors were dissociated with 200U/ml collagenase IV at 37 °C for 30 min. Flow cytometric analysis was carried out on a Canto II Instrument (BD Biosciences).
10
Tumor Infiltrating Immune Cells Analysis
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Mice bearing melanoma allografts were i.p. injected with PBS, VNP20009 and two flagellum-deficient strains. The tumors and spleens were resected after 6 days. Half of the tumor mass was dissected and incubated in PBS containing collagenase I (Gibco), collagenase IV (Sigma–Aldrich), DNase I (Sigma–Aldrich) and hyaluronidase (Worthington) for 1 h at 37 °C. Then the cell suspension was filtered through a 70 μm cell strainer to form single cell suspension. Then red blood cells were removed by 5 min of incubation with red blood cell lysis buffer on ice. The mice spleens were smashed thoroughly between frosted glass slides and filtered through a 70 μm cell strainer to prepare single cell suspension. Then red blood cells were removed by 5 min of incubation with red blood cell lysis buffer on ice.
After that, cells were stained with following antibodies: anti-CD4-PE (#553652, BD Pharmingen), anti-CD8-FITC (#561966, BD Pharmingen), anti-F4/80-PE (#565410, BD Pharmingen) and were processed for flow cytometry analysis by Cytomic FC 500MCL flow cytometer (Beckman Coulter).
After that, cells were stained with following antibodies: anti-CD4-PE (#553652, BD Pharmingen), anti-CD8-FITC (#561966, BD Pharmingen), anti-F4/80-PE (#565410, BD Pharmingen) and were processed for flow cytometry analysis by Cytomic FC 500MCL flow cytometer (Beckman Coulter).
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