Differentiated Monocyte derived macrophages were treated with Accutase for 20 min and detached with gentle pipetting. Cells were washed with FACS buffer (5%FBS, 2mM EDTA, 0.09% sodium azide in PBS) and incubated with FcR binding inhibitor (14-9161-73, ebioscience) for 20min on ice. Cells were then incubated with human TREM-1 phycoerythrin conjugated monoclonal antibody (FAB1278P, R&D System), or mouse IgG1 phycoerythrin Isotype Control (IC002P, R&D System), 30min at 4°C. BD Cytoperm kit (BDB554714, BD Bioscience) was used for intracellular staining of CD68 using human CD68 BV421(BD564943, BD Bioscience) and data was acquired on a Becton Dickinson LSRF Fortessa (Beckman, USA) and analyzed with FlowJo (Tree Star, USA).
Ic002p
Manufactured by R&D Systems
Sourced in United States
The IC002P is a laboratory instrument designed for cell culture applications. It functions as an incubator, providing a controlled environment for cell growth and maintenance. The device is equipped with temperature, humidity, and CO2 regulation capabilities to support optimal conditions for cultivating cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Ic002f, by R&D Systems (3 mentions)
Fab170p, by R&D Systems (2 mentions)
Fab347p, by R&D Systems (2 mentions)
Fab6311p, by R&D Systems (2 mentions)
Ic0041p, by R&D Systems (2 mentions)
Fab1278p, by R&D Systems (1 mentions)
Ab33858, by Abcam (1 mentions)
Sc 7324, by Santa Cruz Biotechnology (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Ab34139, by Abcam (1 mentions)
Facscalibur platform, by BD (1 mentions)
Fab332a, by R&D Systems (1 mentions)
Ic002a, by R&D Systems (1 mentions)
Hsc003, by R&D Systems (1 mentions)
L7914, by Merck Group (1 mentions)
Psih1 h1 copgfp shrna vector, by System Biosciences (1 mentions)
Facscalibur system, by BD (1 mentions)
Anti cd90, by BD (1 mentions)
Ab1791, by Abcam (1 mentions)
Gtx61796, by GeneTex (1 mentions)
10 protocols using ic002p
1
TREM-1 and CD68 Expression in Macrophages
2
Flow Cytometry Characterization of Dental Stem Cells
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For the flow cytometry assay, both hTDSCs and dTDSCs at P3 were used. 1 × 106 cells of each group were incubated with 1 mg of phycoerythrin-conjugated or fluorescein-isothiocyanate-conjugated monoclonal antibodies (R&D Systems) at 4 °C for 1 h. Anti-CD34 (sc-7324; Santa Cruz Biotechnology, Santa Cruz, CA), anti-CD31 (ab33858; Abcam, Cambridge, UK), anti-CD44 (BD550974; BD Bioscience), anti-CD45 (BD559135; BD Bioscience), and anti-CD90 (BD554898; BD Bioscience) were the antibodies used in this study. Phycoerythrin-conjugated or fluorescein-isothiocyanate-conjugated isotype-matched IgG1 were used as negative controls (IC002P or IC002F; R&D Systems). The stained cells were washed with ice-cold PBS containing 2% BSA before analysis using the LSRFortessa flow cytometer (Becton Dickinson, San Jose, CA). About 1 × 104 events were counted for each sample. The percentage of cells with a positive signal was calculated using the WinMDI Version 2.9 program (The Scripps Research Institute, La Jolla, CA).
3
Flow Cytometric Analysis of Stem Cell Markers
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For flow cytometric analysis, the cells were harvested and dissociated into single cells with 0.25% trypsin and then washed in PBS plus 0.5% BSA twice. After washing, the cells were stained with antibodies to CXCR4 (FAB170P; R&D system), c-Kit (FAB332A; R&D system) and DAZL (ab34139; abcam) for 40 min at 4°C. After washing by PBS twice, the cell pellet was re-suspended in 300 μl PBS for the final flow cytometric analysis of CXCR4 and c-Kit. Besides, the second antibody (ZF-0511, 1:400, Alexa Fluor 488-conjugated goat anti-rabbit IgG, ZSGB-BIO) was added into DAZL treatment for 40 min at 4°C. After that, the cells were washed twice with PBS and re-suspended in 300 μl of PBS for the final flow cytometric analysis. As a control for the analysis, the undifferentiated cells in a separate tube were treated with a mouse IgG1 APC-conjugated antibody (IC002A; R&D system) and a mouse IgG1 isotype control-PE (IC002P; R&D system) for c-Kit and CXCR4, respectively. For DAZL, the undifferentiated cells were only strained with the second antibody. Finally, cells analysis was performed on the Becton-Dickinson FACS Calibur platform.
4
Cell Surface Expression of DR4 and DR5
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Cell surface expression of DR4 and DR5 was performed using PE-conjugated antibodies (R&D Systems, FAB347P and FAB6311P). Cells were harvested at 70–80% confluence and were washed twice with PBS containing 1% FBS. Anti-DR4-PE or anti-DR5-PE was then added to a final concentration of 10 µg/mL to a 25 µL cell suspension containing 1 × 105 cells. Control samples were incubated with isotype-matched PE-conjugated control antibodies (R&D Systems, IC002P and IC0041P). Antibody incubation was performed for 1 h at 4 °C in the dark. Cells were then washed twice with PBS containing 1% FBS and resuspended in 0.5 mL of the solution for flow cytometric analysis.
5
Lentiviral Knockdown of IL1RAP in Leukemia Cells
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For knockdown of IL1RAP, we cloned shRNA template oligonucleotides into the pSIH1-H1-copGFP shRNA vector (System Biosciences) and produced lentiviral particles as previously described (Barreyro et al., 2012 (link)). THP-1, HEL, or TF-1 cells were transduced with a nonsilencing control (luciferase) shRNA- or IL1RAP-specific shRNAs (MOI = 1), and transduced (GFP+) cells were FACS sorted before transplantation. For all shRNA transduction experiments, 8 µg/ml polybrene was added to cells before incubation with virus, and spin infection was performed after addition of virus (1,000 rcf for 1 h at 32°C) to facilitate transduction. Knockdown efficiency was measured by flow cytometry using anti–human IL-1RAcP/IL-1R3 APC or PE (FAB676A and FAB676P; R&D) and mouse IgG1 APC (17-4714; eBioscience) or PE (IC002P; R&D) as controls.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.
6
Flow Cytometric Analysis of CD44 and CD90
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Phycoerythrin (PE) or fluorescein isothiocyanate (FITC) (BD Biosciences)-conjugated mouse anti-rat monoclonal anti-CD44 (550974; BD Biosciences) and anti-CD90 (551401; BD Biosciences) were used. PE- or FITC-conjugated isotype-matched IgG1 were used as negative controls (IC002P or IC002F, R&D systems, Inc., Minneapolis, US). TDCs (5 × 105; P1) were incubated with 1 μg of antibodies away from the light for 45 min at room temperature. After washing and centrifugation at 4000× g for 5 min, the cells were resuspended in 300 μL of ice cold PBS (with 10% FBS and 1% sodium azide) and subjected to fluorescence-activated cell sorting (FACS) analysis (BD Biosciences). A BD FACSCalibur™ system (BD Biosciences) was used to calculate the percentage of cells with positive signals.
7
Antibody Utilization for Western Blot, IF, and ChIP
8
Flow Cytometric Analysis of IL-17RA Expression
9
Profiling Human Breast Cancer Cell Lines
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The human breast cancer cell lines including AU565, BT474, MDA-MB-453, HCC1428, MDA-MB-361, T47D, MCF7, ZR751, HCC1500, HCC1937, HCC1954, MDA-MB-468, SKBR3, MDA-MB-157, MDA-MB-231, HCC38, HCC2157, and BT549 were purchased from American Type Culture Collection (ATCC). All cell lines were cultured per ATCC recommendation and were tested for the absence of mycoplasma contamination regularly. Recombinant human TRAIL protein, containing amino acids 114-281 of human TRAIL was produced by E. coli as homotrimers and was purchased from R&D Systems (375-TEC). Antibodies specific to human caspase-3 (8G10), caspase-8 (1C12), DR4 (D9S1R), and DR5 (D4E9) were purchased from Cell Signaling Technology. Keratin 8/18 antibodies were purchased from Cell Signaling (C51) and BioLegend (1E8). GAPDH antibody was purchased from Novus (2D4A7). Horseradish peroxidase–conjugated goat anti-rabbit IgG1 (sc-2054), goat anti-mouse IgG1 (sc-2969), and donkey anti-goat IgG (sc-2020) were purchased from Santa Cruz Biotechnology. Phycoerythrin (PE)-conjugated monoclonal antibodies to DR4 (FAB347P) and DR5 (FAB6311P) and corresponding IgG1 (IC002P) and IgG2b (IC0041P) controls were purchased from R&D Systems.
10
Flow Cytometry Analysis of Cell Surface Markers
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Expression of cell surface markers at the protein level was selectively analyzed by flow cytometry according to the microarray results. Holo-, mero-, and paraclones (n = 3, each) were harvested and suspended in PBS at a concentration of 1 million cells per mL. For the surface staining, anti-hTHSD1 (R&D systems #FAB3715P), with an appropriately matched isotype control mAb (R&D systems #IC002G), was used. The following antibodies were used for intracellular staining: anti-hFAP and anti-hDCBLD2 (R&D systems # FAB3715P, AF6269), with an appropriately matched isotype control mAb (R&D systems # IC002P, 5-001-A). The results were assessed using an FACScan (BD Biosciences, San Jose, CA, USA).
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