For detection of CD40 expression, cells were cultured in flasks until approximately 80% confluent. Alternatively, for treatment with cytokines, cells were seeded in 24-well plates at 5 × 104 cells/well and, after overnight incubation, they were treated with fresh medium containing 103 units/ml TNF-α or IFN-γ (R&D Systems/Bio-Techne) for 48 h. Cells were then harvested, washed and re-suspended in FACS buffer22 (link). CD40 expression was detected using PE-conjugated mouse anti-human CD40 antibody and an isotype control IgG1-PE (BD Biosciences, Berks, UK). In all, 10,000 events were acquired on a Guava EasyCyte flow cytometer and results analysed using EasyCyte software (Millipore, Watford, UK).
Igg1 pe isotype control
Manufactured by BD
Sourced in United Kingdom, United States
The IgG1-PE isotype control is a laboratory reagent used in flow cytometry experiments. It serves as a control to establish background fluorescence levels when analyzing cell samples labeled with antibodies conjugated to the Phycoerythrin (PE) fluorochrome.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by R&D Systems (2 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Easycyte software, by Merck Group (1 mentions)
Pe conjugated mouse anti human cd40 antibody, by BD (1 mentions)
S3 cell sorter, by Bio-Rad (1 mentions)
Cd38 pe, by BD (1 mentions)
Cxcr4 pe, by BD (1 mentions)
Cd44 fitc, by BD (1 mentions)
Vybrant dyecycle green stain, by Thermo Fisher Scientific (1 mentions)
Methylcellulose, by STEMCELL (1 mentions)
Goat anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
β actin, by Merck Group (1 mentions)
Goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Flt3 s 18, by Santa Cruz Biotechnology (1 mentions)
Pmapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
8 protocols using igg1 pe isotype control
1
CD40 Expression Detection Protocol
2
Immunophenotyping and Cell Cycle Analysis
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Cells were stained with CD138 PE-Cy5 (Beckman Coulter), CD38 PE, Isotype Control IgG1 PE, CXCR4 PE, or CD44 FITC (BD Pharmingen), CD74 FITC (Abcam), and Blimp1 Alexa-Fluor 488 (R&D) by standard procedures. All flow cytometry analysis used the BD FACScan, the BioRad S3 Cell Sorter and data was analyzed using CellQuest software. Cells were stained with Vybrant DyeCycle Green Stain (Invitrogen) and cell cycle analysis was performed using CellQuest software. Freshly sorted populations were also resuspended in methylcellulose (Stem Cell Technologies) at 104 cells/ml as per manufacturer’s instructions. Colonies were counted manually and photographs were taken using a light microscope under 4X magnification. Colonies were measured using the annote tab to compare pixel number to the scale created by the Kodak film software.
3
Characterization of FLT3 Mutations
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DNA constructs and vectors were used as described before [9] (link), [25] (link). FLT3-I867S and FLT3-D839G constructs were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). Denotation: W78: ITD1, Npos: ITD2, W51: ITD3. Figure S1 displays the locations and insertions respectively substitutions of the analyzed mutations.
The following antibodies were used: FLT3 (S18) and goat anti-mouse secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). AKT, MAPK, pSTAT5 (Tyr694), pAKT (Ser473) and pMAPK (Thr202/Tyr204) all from Cell Signaling Technology (Danvers, MA). STAT5 from R&D Systems (Minneapolis, MN). β-actin and goat anti-rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO). CD-135-PE from Beckman Coulter (Brea, CA). IgG1 PE Isotype control from BD Pharmingen (BD Bioscience, Franklin Lakes, NJ).
The following antibodies were used: FLT3 (S18) and goat anti-mouse secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA). AKT, MAPK, pSTAT5 (Tyr694), pAKT (Ser473) and pMAPK (Thr202/Tyr204) all from Cell Signaling Technology (Danvers, MA). STAT5 from R&D Systems (Minneapolis, MN). β-actin and goat anti-rabbit secondary antibody from Sigma-Aldrich (St. Louis, MO). CD-135-PE from Beckman Coulter (Brea, CA). IgG1 PE Isotype control from BD Pharmingen (BD Bioscience, Franklin Lakes, NJ).
4
Quantifying Neutrophil Surface Markers
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The cell membrane expression of CD11b, CD63, and CD66b was measured. The samples were incubated for 30 min on ice with one of the following mixes: (A) mouse anti-human phycoerythrin (PE)-conjugated CD63+mouse anti-human FITC-conjugated CD66b, (B) mouse anti-human PE-conjugated CD11b+mouse anti-human FITC-conjugated CD66b, or (C) IgG1-PE isotype control and IgG1-FITC isotype control (all from BD Biosciences, San Diego, CA, USA) at a final concentration of 10 μg·mL−1. The PMNs were gated according to their relative size (forward scatter) and granularity (sideward scatter). The expression of the cell surface markers was calculated as the mean fluorescence intensity (MFI) and corrected for non-specific binding of isotype control antibodies.
5
CD46 Expression on T Cell Subsets
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PBMCs were stained with αCD3-FITC (UCHT1, BD Bioscience), αCD4-Brilliant Violet 421 (RPA-T4, BD Bioscience), αCD8-PC5 (B9.11, Beckmann Coulter), αCD46-PE (MEM-258, Sigma-Aldrich), IgG1-PE isotype control (BD Bioscience), and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies) in the volumes indicated by the manufacturers. CD46 expression was determined on respectively CD4+CD8− and CD4-CD8+ cell populations following gating on first live cells and then CD3+ cells. Isolated and in vitro activated CD8+ and CD4+ T cells were stained with αCD46-PE (MEM-258) and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and the expression of CD46 was determined on the live cells. The isotype control antibody was used to visualize the background PE fluorescence signal. Data were acquired on a NovoCyte flow cytometer (ACEA Bioscience Inc.) and processed in FlowJo (Tree Star). The detailed gating strategy for all flow cytometry experiments is presented as supplemental figurers (Fig. 1S–4S). Fluorescent data was displayed using bi-exponential visualization according to best practice [14 (link)].
6
Modulating Immune Signaling Pathways
7
Flow Cytometric Profiling of Skeletal Muscle Stem/Progenitor Cells
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Flow cytometry was conducted using the skeletal muscle supernatant. Specific antibodies were used for cell labelling: CD34 antibody for stem/progenitor cells, CD44 and CD105 antibodies for MSCs, and CD45 antibody for haematopoietic lineage cells (all from BD Biosciences, Franklin Lakes, New Jersey). Samples underwent regular flow cytometric profiling with a FACSCalibur Analyser and CellQuest Pro Software (BD Biosciences Immunocytometry Systems, San Jose, California). Dead cells were excluded from the plots on the basis of propidium iodide (PI) staining (Sigma-Aldrich Corp., St Louis, Missouri). Cells were washed twice with Hanks’ balanced salt solution (HBSS) containing 3.0% heat-inactivated foetal bovine serum (FBS) and incubated with monoclonal antibodies for 30 minutes at 4°C after Fc recepter blocking reagent (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The stained cells were washed three times with HBSS/3.0% FBS, resuspended in 0.5 mL of HBSS/3.0% FBS/PI, and analysed. The following monoclonal antibodies were used to identify the CD34+/CD45dim subpopulation (the stem/progenitor cell fraction), and the CD44+/CD105+ subpopulation (the MSC fraction): CD34-FITC (clone RAM34), CD44-APC (clone G44–26), CD105-PE (clone 266), CD45-FITC (clone HI30), IgG1-PE isotype control, and IgG1-FITC (all from BD Biosciences Pharmingen San Diego, California).
8
Quantifying HER-2/neu Expression
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Cells were harvested after 24 h under normoxia or hypoxia and stained with an anti-HER-2/neu-PE (BD) antibody or with the IgG1-PE isotype control (BD), respectively, for 20 min as recently described [47 (link)]. Fluorescence intensity was determined by flow cytometry (FACSCalibur, BD). The results were expressed as mean specific fluorescence intensity (MFI). Three independent experiments were performed.
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