The levels of the CD44+, CD24 -, and CD133+cells in each group were evaluated using flow cytometry with a fluorescein isothiocyanate (FITC)-conjugated CD44 antibody (cat. no. 347943; BD Biosciences), phycoerythrin (PE)-conjugated CD24 antibody (cat. no. 555428; BD Biosciences), and a PE-conjugated CD133 antibody (cat. no. 12-1331-82; eBioscience; Thermo Fisher Scientific, Inc.). Mouse immunoglobulin (Ig)G2a-FITC (cat. no. 555573; BD Biosciences), mouse IgG2a-PE (cat. no. 554648; BD Biosciences), and mouse IgG1K-PE (cat. no. 12-4714-42; eBiosciences Thermo Fisher Scientific, Inc.) were used as the isotype controls (all of antibodies diluted at 1:100 ratio). Cells were incubated with antibodies at 4˚C for 45 minutes and analyzed using FACSCaliburTM (Becton Dickinson). The raw data were analyzed using Flowing Software version 2.5.0 (Perttu Terho).
Mouse igg2a pe
Manufactured by BD
Sourced in United States
Mouse IgG2a-PE is a fluorescently-labeled antibody that binds to the IgG2a isotype of mouse immunoglobulin. It can be used for the detection and quantification of mouse IgG2a-positive cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg1 fitc, by BD (3 mentions)
Pe mouse igg1, by BD (2 mentions)
Apc mouse igg1, by BD (2 mentions)
Fitc mouse anti human cd4, by BD (2 mentions)
Pe conjugated cd133 antibody, by BD (1 mentions)
Facscaliburtm, by BD (1 mentions)
Mouse igg1k pe, by BD (1 mentions)
Anti cd31 pe, by BD (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Anti cd90 apc, by BD (1 mentions)
Anti cd105 pe, by BD (1 mentions)
Anti cd106 pe, by BD (1 mentions)
Anti cd45 apc, by BD (1 mentions)
Anti cd36 pe, by BD (1 mentions)
Anti hla dr pe, by BD (1 mentions)
Anti cd73 pe, by BD (1 mentions)
Anti cd54 apc, by BD (1 mentions)
Software, by FlowJo (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd133 apc, by Miltenyi Biotec (1 mentions)
8 protocols using mouse igg2a pe
1
Quantifying Cancer Stem Cell Markers
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The levels of the CD44+, CD24 -, and CD133+cells in each group were evaluated using flow cytometry with a fluorescein isothiocyanate (FITC)-conjugated CD44 antibody (cat. no. 347943; BD Biosciences), phycoerythrin (PE)-conjugated CD24 antibody (cat. no. 555428; BD Biosciences), and a PE-conjugated CD133 antibody (cat. no. 12-1331-82; eBioscience; Thermo Fisher Scientific, Inc.). Mouse immunoglobulin (Ig)G2a-FITC (cat. no. 555573; BD Biosciences), mouse IgG2a-PE (cat. no. 554648; BD Biosciences), and mouse IgG1K-PE (cat. no. 12-4714-42; eBiosciences Thermo Fisher Scientific, Inc.) were used as the isotype controls (all of antibodies diluted at 1:100 ratio). Cells were incubated with antibodies at 4˚C for 45 minutes and analyzed using FACSCaliburTM (Becton Dickinson). The raw data were analyzed using Flowing Software version 2.5.0 (Perttu Terho).
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The levels of the CD44+, CD24 -, and CD133+cells in each group were evaluated using flow cytometry with a fluorescein isothiocyanate (FITC)-conjugated CD44 antibody (cat. no. 347943; BD Biosciences), phycoerythrin (PE)-conjugated CD24 antibody (cat. no. 555428; BD Biosciences), and a PE-conjugated CD133 antibody (cat. no. 12-1331-82; eBioscience; Thermo Fisher Scientific, Inc.). Mouse immunoglobulin (Ig)G2a-FITC (cat. no. 555573; BD Biosciences), mouse IgG2a-PE (cat. no. 554648; BD Biosciences), and mouse IgG1K-PE (cat. no. 12-4714-42; eBiosciences Thermo Fisher Scientific, Inc.) were used as the isotype controls (all of antibodies diluted at 1:100 ratio). Cells were incubated with antibodies at 4˚C for 45 minutes and analyzed using FACSCaliburTM (Becton Dickinson). The raw data were analyzed using Flowing Software version 2.5.0 (Perttu Terho).
2
Immunophenotyping of Mesenchymal Cell Types
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The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described [13 (link)]. The cells grown to 60% confluence were suspended at a density of 5 × 105 cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 104 events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.
3
Enumeration of Circulating Endothelial Cells
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Whole blood staining's were performed within 24 hours after blood collection. White blood cells were counted using Cell-Dyn Sapphire Hematology System (Abbott Diagnostics, Chicago, IL, USA). For CEC and CEP staining 5 millions cells per tube were used, while for monocyte and isotype staining's 1 million cells was used. Directly labeled antibodies were added on total blood and incubated for 20 minutes at 4°C, followed by 10 minutes red-blood-cells lysis (Bühlmann Laboratories, Schönenbuch, Switzerland) and washing using cold PBS.
All anti-human antibodies were used at the concentration give in manufacturer's instructions of use: anti-CD45-V450, anti-CD146-Pe, anti-CD31-AlexaFluo 647, anti-CD34-PeCy7, anti-VEGFR2-Pe, anti-CD11b-V450, anti-CD64-FITC, anti-JAM1-Pe, anti-TIE2-AlexaFluo 647, anti-KIT-PeCy7, anti-CD195-Pe, mouse IgG2a-AlexaFluor 647, mouse IgG1-Pe, mouse IgG1-PeCy7, mouse IgG1-APC, mouse IgG2a-Pe, 7AAD (all from Becton Dickinson), Syto16 (Life Technologies, Carlsbad, CA, USA), anti-CD133-APC (Miltenyi), anti-VEGFR1-APC (R & D Biosystems, Minneapolis, MN, USA).
BD FACSCanto II (Becton Dickinson) instrument was used to perform experiments and FlowJo 9.8.3 (Treestar Inc., Ashland, OR, USA) software was used to analyze all data.
All anti-human antibodies were used at the concentration give in manufacturer's instructions of use: anti-CD45-V450, anti-CD146-Pe, anti-CD31-AlexaFluo 647, anti-CD34-PeCy7, anti-VEGFR2-Pe, anti-CD11b-V450, anti-CD64-FITC, anti-JAM1-Pe, anti-TIE2-AlexaFluo 647, anti-KIT-PeCy7, anti-CD195-Pe, mouse IgG2a-AlexaFluor 647, mouse IgG1-Pe, mouse IgG1-PeCy7, mouse IgG1-APC, mouse IgG2a-Pe, 7AAD (all from Becton Dickinson), Syto16 (Life Technologies, Carlsbad, CA, USA), anti-CD133-APC (Miltenyi), anti-VEGFR1-APC (R & D Biosystems, Minneapolis, MN, USA).
BD FACSCanto II (Becton Dickinson) instrument was used to perform experiments and FlowJo 9.8.3 (Treestar Inc., Ashland, OR, USA) software was used to analyze all data.
4
Signaling Pathway Regulation in Cells
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Dimethyl sulfoxide (DMSO), cycloheximide (CHX), N-acetyl-L-cysteine (NAC), fumonisin B1, desipramine and GW4869 were purchased from Sigma-Aldrich (St Louis, MO, USA). The fluorescence-labeled monoclonal antibody (mAb) anti-human TLR2-phycoerythrin (TLR2-PE) and TLR4-PE were purchased from eBioscience (San Diego, CA, USA). The mouse IgG2a-PE was purchased from Becton Dickinson (San Jose, CA, USA). Stress-activated protein kinases (SAPK)/c-Jun N-terminal kinases (JNK) rabbit Ab (#9252), phospho-SAPK/JNK (Thr183/Tyr185) mouse mAb (#9255), p44/42 mitogen–activated protein kinase (MAPK) (extracellular signal-regulated kinase (ERK)1/2) rabbit mAb (#4695), phospho-p44/42 MAPK (Thr202/Tyr204) rabbit mAb (#4370), apoptosis signal–regulating kinase 1 (ASK1) rabbit Ab (#3762), phospho-ASK1 (Thr845) rabbit Ab, phospho-c-Jun (Ser63) rabbit mAb (#2361), phopho-MAPK kinase 7 (MKK7) (Ser271/Thr275) rabbit Ab (#4171), β-actin Ab (#4967), anti-rabbit IgG horseradish peroxidase (HRP)-linked Ab (#7074) and anti-mouse IgG HRP-linked Ab (#7076) were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). The fluorescent probe 3′-(p-hydroxyphenyl) fluorescein (HPF) was obtained from Molecular Probes (Eugene, OR, USA).
5
TLR2/TLR6 Signaling Pathway Activation
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LPS (Escherichia coli 055:B5) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St Louis, MO, USA). The TLR2/TLR6 agonist FSL-1 was purchased from InvivoGen (San Diego, CA, USA). The fluorescence-labeled monoclonal antibodies (mAbs), anti-human TLR2-phycoerythrin (TLR2-PE) and TLR4-PE were purchased from eBioscience (San Diego, CA, USA). Anti-human tumor necrosis factor-α-fluorescein isothiocyanate (TNF-α-FITC) and mouse IgG2a-PE were purchased from Becton Dickinson (San Jose, CA, USA). Mouse IgG1-FITC was purchased from Beckman Coulter (Fullerton, CA, USA). Phospho-SAPK/c-Jun N-terminal kinase (JNK) (Thr183/Tyr185) mouse mAb, phospho-p38 mitogen-activated protein kinase (MAPK; Thr180/Tyr182) rabbit mAb, phospho-p44/42 MAPK [Extracellular signal-regulated kinase (ERK)1/2; Thr202/Tyr204] rabbit mAb, and Alexa Fluor® 488-conjugated goat anti-rabbit IgG were purchased from Cell Signaling Technology Japan, KK (Tokyo, Japan). Alexa Fluor® 488-conjugated goat anti-mouse IgG was purchased from Invitrogen (Carlsbad, CA, USA). SP600125 and PD98059 were purchased from Sigma-Aldrich, and SB203580 was obtained from Calbiochem (San Diego, CA, USA).
6
Nanoparticle Synthesis and Characterization
7
Doxorubicin-Resveratrol Nanoparticle Synthesis
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Doxorubicin hydrochloride (Dox·Hcl) was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, China). Resveratrol (Res) was bought from Guan Jie Biotech (Xian, China). 1-Bromo-4-chlorobutane and triphenylphosphine (TPP) were obtained from Aladdin Co., (Shanghai, China). K2CO3 and NaI were supplied by Sinopharm Chemical Reagent Co., (Shanghai, China). Dioleoylphosphatidylethanolamine (DOPE) and hydrogenated soybean phospholipids (HSPC) were purchased from Shanghai A.V.T Technology Co., Ltd. (Shanghai, China). Cholesterol and cholesteryl hemisuccinate (CHEMS) were bought from J&K Scientific Co., Ltd. (Beijing, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), cell mitochondria isolation kit, mitochondrial membrane potential assay kit with JC-1, Caspase 3,8,9 assay kit, ATP assay kit, Lysotracker Green DND and Hoechst 33258 were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). FITC anti-human CD44, FITC Mouse IgG1, PE anti-human CD24 and PE Mouse IgG2a were bought from BD Biosciences (Franklin Lakes, NJ, USA). β-Actin, β-catenin and cyclin D1 primary antibodies and corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were provided by Abcam (Cambridge, UK).
8
Antibody Detection and Cytotoxicity Assays
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The following antibodies were used: Alexa Fluor 647–coupled anti-GFP (catalog no. 565197, BD Pharmingen); Alexa Fluor 647 rat immunoglobulin G2a (IgG2a), κ isotype control (catalog no. 400526); Alexa Fluor 647 anti-human CD195 (catalog no. 313712) from BioLegend; phycoerythrin (PE) mouse IgG1, κ isotype control (catalog no. 550617); PE mouse IgG2a, κ isotype control (catalog no. 553457); fluorescein isothiocyanate (FITC) mouse IgG2a, κ isotype control (catalog no. 555573); FITC mouse IgG1 κ isotype control (catalog no. 555748); PE mouse anti-human CD184 (catalog no. 555974); FITC mouse anti-human CD4 (catalog no. 555346); FITC mouse anti-human CD3 (catalog no. 555332); and PE mouse anti-human CD11b/Mac1 (catalog no. 555388) from BD Biosciences.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
The amount of lactate dehydrogenase (LDH) released into supernatants was quantified using a Pierce LDH Cytotoxicity Assay kit (catalog no. 88953, Thermo Fisher Scientific). The Alliance HIV-1 p24 ELISA (enzyme-linked immunosorbent assay) Kit (catalog no. NEK050, PerkinElmer) was used to determine the amount of capsid p24 protein in supernatants and cell lysates. LDH and p24 quantification were performed following the manufacturer’s instructions.
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