Surface expression of IL-12Rβ2 and intracellular T-bet were assayed at 24, 48, 72, and 96 hours as previously described (3 (link)). Briefly, splenocytes were surface stained with fluorochrome-conjugated anti-CD4 (GK1.5) (BD Biosciences), -IL-12Rβ2 (305719), and -mouse IgG1 (R&D Biosystems) Abs, fixed in BD Cytofix/Cytoperm buffer, washed in BD Perm/Wash buffer, and then stained with anti-T-bet (O4-46) Ab (BD Biosciences) and Mouse IgG1,κ according to manufacturer’s guidelines. Cells were acquired on a FACSCalibur and analyzed with FlowJo version 9.6.1 (Tree Star Incorporated). Pooled data for T-bet and IL-12Rβ2 percentages were calculated from at least 4 independent experiments.
Mouse igg1
Manufactured by R&D Systems
Sourced in United States, Germany
Mouse IgG1 is a purified immunoglobulin G1 (IgG1) isotype antibody derived from mouse. IgG1 is one of the four subclasses of IgG, the most abundant class of antibodies in the human body. The core function of Mouse IgG1 is to serve as a research tool for the study of IgG1 properties and interactions.
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Lab products found in correlation
Rat igg2a, by R&D Systems (3 mentions)
Mouse igg1, by BD (2 mentions)
Mouse igg2b, by R&D Systems (2 mentions)
Fortressa, by BD (2 mentions)
Mouse anti il 10r blocking antibody, by R&D Systems (2 mentions)
Mouse anti tnfα receptors 1 and 2, by R&D Systems (2 mentions)
Cd14 microbead, by Miltenyi Biotec (2 mentions)
Adg72, by Sekisui (2 mentions)
Anti hla dr, by BD (2 mentions)
Anti cd45, by BD (2 mentions)
Cytofix cytoperm buffer, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Ds fi2 300 megapixel digital camera, by Nikon (1 mentions)
Hematoxylin solution, by Merck Group (1 mentions)
Anti ifn γr1, by R&D Systems (1 mentions)
Anti cd13, by Abcam (1 mentions)
Mouse igg2bκ, by BioLegend (1 mentions)
Facscalibur, by BD (1 mentions)
Mouse igg1k, by BD (1 mentions)
Anti nkg2d antibody, by R&D Systems (1 mentions)
22 protocols using mouse igg1
1
Temporal Expression of IL-12Rβ2 and T-bet
2
Immunohistochemical Analysis of Bone Markers
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Samples were stained using hematoxylin solution (Mayers; Sigma Aldrich, St Louis, Missouri, United States). Immunohistochemical staining using ALP rabbit antihuman monoclonal antibody LS-B6663 (LifeSpan Biosciences Inc., Seattle, United States), rabbit anticollagen I polyclonal antibody (BIOSS, United States), and osteocalcin monoclonal antibody mouse IgG1 (Clone #190125; R&D Systems, United States) was also conducted. Microscopic observation was performed using a light microscope (Nikon H600 L, Tokyo, Japan) equipped with a DS-Fi2 300-megapixel digital camera and Nikon Image System picture editing software. Data of ALP, type 1 collagen, and osteocalcin expression were calculated using a Remelle for immunohistochemistry index scale from five different fields of view at ×400 magnification. Extension of the trabecular bone area was studied at × 200 magnification.
3
Surface Protein Staining and Flow Cytometry
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For staining of surface proteins, 1 × 105 cells were seeded for drug treatment and detached on day 3 after seeding (resulting in ∼5 × 105 cells). Cells were resuspended in ice-cold PBS with 3% bovine serum albumin (PBS 3% BSA) containing a primary antibody at the individual working dilution. After 1 h incubation at 4 °C, cells were washed twice and taken up in ice-cold PBS 3% BSA containing a goat-anti-mouse antibody labeled with phycoerythrin (PE) (Santa Cruz, Dallas, USA). After incubation for 1 h at 4 °C in the dark, cells were washed twice, resuspended in ice-cold PBS and analyzed on a BD FACSCalibur (BD biosciences, New Jersey, USA). All washing steps were performed with ice-cold PBS. Data were analyzed using FlowJo Software (Tree Star, USA). Primary antibodies and respective concentrations used during this study were anti-IFN-γR1 (1 μg/ml; R&D systems, Minneapolis, Canada), anti-CD13 (1 μg/ml; abcam), anti-HLA-A∗02 (1 μg/ml; abcam, Cambridge, UK), anti-β2 microglobulin (1 μg/ml) and anti-HLA-A, B, C (W6/32; 4 μg/ml) (both Biolegend, London, UK). Isotype control antibodies were mouse IgG2bκ (Biolegend) and mouse IgG1 (R&D systems, Wiesbaden, Germany). For each condition data of at least 10,000 cells were collected. Staining for intracellular IFN-γ and surface CD8 was performed as described previously [9] (link).
4
Characterizing Cancer Stem Cells by Immunofluorescence
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Sphere-derived cells were proven to be CSC by immunofluorescence characterization for stemness membrane markers. Briefly, tumor spheres were mechanically and enzymatically dissociated to obtain single cells. Cells were fixed and permeablized as previously reported [25 (link)]. Cytospun cells were washed in PBS and exposed overnight at 4°C to antibodies against CD133 (AC133, mouse IgG1, Miltenyi), CD166 (mouse IgG1, R&D Systems), OCT3/4 (sc-5279, mouse IgG2b, Santa Cruz), ALDH-1 (mouse IgG1, BD Bioscience), CD90 (mouse IgG1k, BD Bioscience), or isotype-matched controls. Afterwards, cells were labelled with fluorescein isothiocyanate (FITC) conjugated mouse antibodies (Invitrogen) and nuclei were counterstained using Toto-3 iodide. The fluorescent staining was evaluated with a confocal microscope.
5
NKG2D Receptor Blocking Assay
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This blocking assay was based on methods previously reported (15 (link)). HES-fractionated BM cells from HDs and patients with FA were divided into 2 equal aliquots (range: 0.5 × 106 to 1 × 106 cells) and resuspended in 200 μL IMDM supplemented with 20% FBS and loaded into V-bottomed 96 MicroWell plates (Nunc). One of the aliquots was incubated with mouse IgG1 (25 μg/mL) as an isotype control (clone 11711), and the other one was incubated with the anti-NKG2D antibody (25 μg/mL) to block the NKG2D-activating receptor (clone 149810), both from R&D Systems. Cells were incubated for 10 minutes at RT and resuspended after 5 minutes. Subsequently, cells were gently centrifuged at 60g for 3 minutes to facilitate cell interaction at the well bottom and were then further incubated at 37°C in 5% CO2 for 4 hours. Cells were then collected, resuspended, and seeded in methylcellulose cultures (see experimental scheme in Figure 6 ). Colony numbers generated in the NKG2D-blocking group were normalized with respect to numbers corresponding to the isotype control group. Some experiments were conducted using purified CD56+ cells (effectors cells) and purified CD34+ cells (target cells) sorted by FACS (see above). In these experiments, a ratio of 10:1 (effector/target cells) was used.
6
Cell Co-culture and Antibody Neutralization
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Cell co-culture and flow cytometry analysis were performed as described in the Supplementary Information . The neutralization of anti-IL-10 antibody (R&D, final concentration of 5 μg/ml) and isotype control antibody (R&D, mouse IgG2B) or anti-TGF-β1 neutralizing antibody (R&D, final concentration of 4 μg/ml) and isotype control antibody (R&D, mouse IgG1) was performed during co-culture experiment.
7
Gene Expression Analysis and Validation
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Gene array .cel files were processed using Genespring (GX version 10.01 2100; Agilent), and data analysis was performed using AltAnalyze. Summarization and normalization of the gene array cel. files was performed using the Robust MultiArray Average (RMA) method. Gene expression levels (Ensembl genes) and GO annotation with GO-Elite software were carried out, using default options. Genes differentially expressed were identified using a combination of a >2-fold change in expression and a significance of P < 0.05 (Lima’s, Student’s t-test). Validation was performed using qRT-PCR on a real-time PCR system (HT7900 Fast Real-Time PCR; Applied Biosystems) using GAPDH as a reference endogenous control. Western blot validation was performed as previously described11 (link) using the following antibodies: IL-6, Mouse IgG1 (R&D systems); Akt, p-Akt (Ser473) (D9E) XP, Stat3 (124H6), p-Stat3 (Tyr705) (D3A7) XP® (Cell Signalling). IL-6 secretion was validated using the Human IL-6 Quantitative ELISA Kit (R&D Systems) according to the manufacturers protocol.
8
Immunohistochemical Staining of Rhabdomyosarcomas
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Serial cryostat sections of rhabomyosarcomas were cut to 6 µm, fixed in ice-cold acetone for 10 min, and incubated with the primary Ab or with an isotype control of irrelevant specificity (mouse IgG1; R&D Systems Wiesbaden-Nordenstadt, Germany) at 4°C overnight. Primary Abs were detected with a biotinylated rabbit anti-mouse-Ig or goat-anti-rabbit-Ig (Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by incubation with a peroxidase-labelled avidin-biotin-complex (Vector) for 1 h. The enzyme was visualized with 3, 3′-diaminobenzidine (DAB; Sigma).
9
Multicolor Flow Cytometry Analysis of eMSC Markers
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The coexpression of eMSC markers, CD140b and CD146 on endometrial stromal cells after 15 days of culture was analysed using multi-colour flow cytometry as described [19 ]. Endometrial cells were labelled with phycoerythrin (PE)-conjugated antibody against CD140b (2.5 μg/ml, mouse IgG1, R & D Systems) and fluorescein isothiocyanate (FITC)-conjugated anti-CD146 antibody (1 mg/ml, mouse IgG1, Thermo Fisher Scientific, Waltham, MA USA) or isotype-matched controls. Flow cytometry analysis was performed using a BD Fortressa (BD Biosciences, San Jose, CA, USA) and the FlowJo software (Tree Star, Ashland, OR, USA) at the Centre for PanorOmic Sciences (CPOS) Imaging and Flow Cytometry Core, The University of Hong Kong.
10
Membrane Expression of EGFR, HER2-4 in Normoxia and Hypoxia
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The cellular membrane expression level of EGFR, HER2, HER3, and HER4 under both normoxia and hypoxia was assessed using flow cytometry. Cells were fixed in 4% formaldehyde (10 min) under normoxia or hypoxia after EGFR, HER2, HER3, and HER4 PE‐conjugated antibody incubation (10 μL/106 cells, 25 μg antibody in 1 mL, R&D Systems, Minneapolis, MN, USA). These receptor‐specific antibodies recognize and bind to the extracellular domain of the receptor. Corresponding isotype controls (respectively, rat IgG2A, mouse IgG2B, mouse IgG1 and mouse IgG2A, 10 μL/106 cells, 50 μg antibody in 2 mL; R&D Systems) were included for all samples and served as negative controls. Dead cells were excluded from the analysis by staining with Live/Dead Fixable Far‐Red Dead Cell Stain Kit (Thermo Fisher Scientific, Merelbeke, Belgium). All samples were measured on a FACScan flow cytometer (BD Biosciences, Erembodegem, Belgium). Flow cytometric data were analyzed using flowjo v10.1 (TreeStar Inc., Ashland, OR, USA). The percentage of EGFR‐, HER2‐, HER3‐ and HER4‐positive cells (overton) was determined in comparison with the corresponding isotype control. Furthermore, the signal for aspecific binding was subtracted from the mean fluorescence intensities (=ΔMFI). This parameter indicates the amount of cellular membrane expression of EGFR, HER2, HER3, and HER4 on individual cells.
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