The expression of the myelomonocytic antigens, cluster of differentiation (CD)11b, CD14, CD13 and CD33, on the cell surface was determined by direct immunofluorescence staining and flow cytometry. Briefly, the cells were collected and washed with phosphate-buffered saline (Beyotime). A total of 5×105 cells were stained with the following conjugated antibodies: CD11b-phycoerythrin (PE; catalogue number: 555388), CD14-fluorescein isothiocyanate (FITC; catalogue number: 555397), CD13-PE and CD33-FITC (catalogue numbers 555394 and 555626, respectively, BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated for 15 min at 4°C and then analyzed using a flow cytometer (FACScabilur; BD Biosciences). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit (Beyotime) according the manufacturer’s instructions, and early apoptosis was evaluated by cytofluorometry (FACScabilur, BD Biosciences).
Cd33 fitc
Manufactured by BD
Sourced in United States
CD33-FITC is a fluorescently-labeled antibody that binds to the CD33 antigen, which is expressed on the surface of myeloid cells, including monocytes, granulocytes, and some progenitor cells. The FITC (fluorescein isothiocyanate) label allows for the detection and identification of CD33-positive cells using flow cytometry or other fluorescence-based detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Cd13 pe, by BD (2 mentions)
Cd11b apc, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Phosphate buffered saline (pbs), by Beyotime (1 mentions)
Cd11b phycoerythrin, by BD (1 mentions)
Cd14 fluorescein isothiocyanate fitc, by BD (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Beyotime (1 mentions)
Facscabilur, by BD (1 mentions)
3 3 diaminobenzidine tetrahydrochloride, by Agilent Technologies (1 mentions)
Cd19 apc, by BD (1 mentions)
Cd138 pe, by BD (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Hla dr v450, by BD (1 mentions)
Anti human cd45 apc, by BD (1 mentions)
Gr 1 v450, by BD (1 mentions)
Cd8a v450, by BD (1 mentions)
F4 80 apc cy7, by BD (1 mentions)
Cd4 pe, by BD (1 mentions)
Cd3 fitc, by BD (1 mentions)
Cd25 apc cy7, by BD (1 mentions)
7 protocols using cd33 fitc
1
Characterization of Myelomonocytic Antigens
2
Isolation and Immunophenotyping of Blood Cells
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Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (for myeloblasts from AML patients) or after lysis of red blood cells (for monocytes and neutrophils from patients with non-hematopoietic neoplasms and healthy individuals), washed, stained with monoclonal antibodies CD19-APC, CD33-FITC and CD138-PE (BD Pharmingen, San Diego, USA), and sorted using a flow cytometer [16 (link)].
Indirect immunocytochemical staining was performed by incubating cytospin slides of AML cell lines with monoclonal anti-human IgK at 37°C for 45 minutes. After washing, the slides were incubated with anti-mouse IgG-horseradish peroxidase (Dako, Carpinteria, USA) at room temperature for 20 minutes, washed, and bound antibodies were detected using 3,3”-diaminobenzidine tetrahydrochloride (Dako) [16 (link)].
Indirect immunocytochemical staining was performed by incubating cytospin slides of AML cell lines with monoclonal anti-human IgK at 37°C for 45 minutes. After washing, the slides were incubated with anti-mouse IgG-horseradish peroxidase (Dako, Carpinteria, USA) at room temperature for 20 minutes, washed, and bound antibodies were detected using 3,3”-diaminobenzidine tetrahydrochloride (Dako) [16 (link)].
3
Isolation and Characterization of Tumor and Immune Cells
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The orthotopic HCC tumor tissues or subcutaneous xenografts were dissected out and minced. Then tissues were digested with 0.8 mg/mL Collagenase IV (Sigma, USA) at 37 ˚C for 1 h. The cell suspensions were filtered through 70 μm strainer and resuspended in 36% Percoll (GE Healthcare, UK). PBMCs of anonymous human healthy donors were isolated by Ficoll reagent (Sigma, USA) according to manufacturers’ instructions. For cell surface staining, 1 × 106 cells were incubated with anti-Fc receptor blocking antibody (2.4G2) at 4 ˚C for 15 min. Murine samples were stained with anti-mouse CD45 APC, CD3 FITC, Gr-1 V450, CD4 PE, CD8a V450, CD25 APC-CY7, Ly6C FITC, Ly6G PECY7, CD11 PE, and F4/80 APC-CY7 from BD Bioscience (USA). Human samples were stained with anti-human CD45 APC, CD11 PE, CD33 FITC, and HLA-DR V450 from BD Bioscience (USA). For intracellular staining of Arg1, Foxp3, and Ki67, cells were fixed and permeabilized by Fixation/Permeabilization solution (BD Biosciences, USA) at 4 ˚C for 15 min. Then cells were washed and stained with anti-mouse Arg1, anti-mouse Foxp3, and anti-Ki67 from BD Bioscience (USA). Flow cytometry was performed on a B.D. Influx cell sorter (BD Bioscience, USA). Flowjo software was used to analyze the data.
4
Tumor Dissociation and Flow Cytometry Analysis
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Tumors were minced into small (1–2 mm3) pieces, and digested using a Tumor Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. The resulting supernatant was filtered through a 200-mesh sieve and washed twice with ice-cold PBS12 . healthy donors were incubated for 5 min with Fc-block (BD Biosciences). The phenotype of MDSCs in patients was determined by a combination of surface markers including HLADR-PerCP-Cy5.5 (Clone L243, BD Biosciences), CD33-FITC (Clone P67.6, BD Biosciences), CD11b-APC (Clone CBRM1/5, BD Biosciences), and CD45-BV421 (Clone HI30, BD Biosciences). Afterward, PD-L1 membrane expression was evaluated using PD-L1 (CD274)-PE (Clone 29E.2A3, BD Biosciences).
For the mouse model, single-cell suspensions were stained with either a myeloid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD11b-APC-Cy7 (Clone M1/70, BD Biosciences), Gr-1-PE (Clone RB6-8C5, BD Biosciences), and PD-L1-APC (Clone 10F.9G2, BD Biosciences) or a lymphoid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD4-PerCP/Cy5.5 (Clone RM4-5, BD Biosciences), CD8-PE (Clone 53–6.7, BD Biosciences), CD62L-APC (Clone MEL-14, BD Biosciences), and CD107a-APC (Clone 1D4B, BD Biosciences). Flow cytometry was performed with a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (TreeStar, Inc, Ashland, OR).
For the mouse model, single-cell suspensions were stained with either a myeloid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD11b-APC-Cy7 (Clone M1/70, BD Biosciences), Gr-1-PE (Clone RB6-8C5, BD Biosciences), and PD-L1-APC (Clone 10F.9G2, BD Biosciences) or a lymphoid panel of antibodies comprising CD45-FITC (Clone 30-F11, BD Biosciences), CD4-PerCP/Cy5.5 (Clone RM4-5, BD Biosciences), CD8-PE (Clone 53–6.7, BD Biosciences), CD62L-APC (Clone MEL-14, BD Biosciences), and CD107a-APC (Clone 1D4B, BD Biosciences). Flow cytometry was performed with a BD FACS Canto II flow cytometer. Data were analyzed using FlowJo software (TreeStar, Inc, Ashland, OR).
5
Flow Cytometry Phenotyping of Expanded Colonies
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Colonies expanded from blood were trypsinised, washed and stained with antibodies: CD31-FITC, CD90-PE, CD105-PE (AbD Serotec, Kidlington, UK), CD19-PE, CD33-FITC, CD34-PerCp, CD45-PE-Cy7, CD61-FITC, CD73-PE, (BD Biosciences, Oxford, UK) and CD271-APC (Miltenyi Biotec), at manufacturers recommended concentrations. Cells were washed, 30,000 events captured on a LSRII flow cytometer and the data analysed using FACSDiva Software (both BD Biosciences).
6
Multicolor Flow Cytometry Analysis of PBMC
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PBMC samples were thawed in 37°C RPMI medium 1640 + GlutaMAX (Life Technologies) and thereafter washed in RPMI and stained in PBS containing 0.5% bovine serum albumin. For phenotyping of CD3+ T-cells, the following antibodies were used: CD45RA-FITC, CD62L-PE, CCR7-PE-CY7, CD3-APC, CD8-BV421, CD4-HV510 (BD Biosciences), CD27-PerCP (Nordic Biosite). Natural Killer cells, B-cells, and γ/δ cells were stained with the following antibodies: CD16-FITC, CD56-PE, CD19-PE-CY7, CD3-APC (BD Biosciences), and γ/δ -BV421 (Nordic Biosite). Myeloid derived suppressor cells were stained with: CD33-FITC, HLA-DR-PerCP, lineage = CD3-, CD19-, and CD56-PE-Cy7, CD11b-APC (BD Biosciences), CD14-BV421 (Nordic Biosite). Regulatory T-cells were stained with CD45RA-FITC, CCR4-PerCP-Cy5.5, CD127-PE-Cy7, CD4-APC, CD25-BV421 (BD Biosciences), FoxP3-PE (eBiosciences). Dead cell marker APC-Cy7 near IR (Invitrogen) fluorescent reactive dye was used to exclude dead cells. For intracellular staining of transcription factor FoxP3, we used Transcription Factor Staining Buffer set (eBioscience) according to guidelines issued by the manufacturer.
7
Multi-parameter Flow Cytometry Analysis
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FACS was performed on an LSR II flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (TreeStar). T cells were stained with the following conjugated antibodies: CD4 FITC (BD/555346), CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD8 AF700 (Invitrogen/MHCD0829), murine TCR-β (mTCR-β) APC (BD/553174), and pMHC-multimers PE. Target cells with transduced WT1 or HLA-alleles were stained with: NGF-R/CD271 APC (Sanbio/CL10013APC), CD34 APC (BD/555824), murine CD19 PE (BD/557399), and HLA-A2 PE (BD/558570). Non-malignant hematopoietic subsets with: CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD34 APC (BD/555824), CD80 PE (BD/557227), and CD86 PE (BD/555658). AML samples with: CD13 PE (BD/347406), CD33 FITC (BD/555626), and CD34 APC (BD/555824).
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