Igg1 pe

Manufactured by Thermo Fisher Scientific

Sourced in United States

IgG1-PE is a fluorescently labeled immunoglobulin G1 (IgG1) antibody conjugate. It is designed for use in flow cytometry applications to detect and quantify target cells or molecules.

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Rat IgG1-PE (2 citations) Anti-mouse IgG1-PE (2 citations) PE-conjugated mouse IgG1 (4 citations) PE mouse IgG1 (P3.6.2.8.1, (2 citations) Mouse IgG1 K Isotype Control PE (9 citations) PE)-conjugated mouse IgG1 K immunoglobulin isotype control (4 citations) PE rat IgG1 κ isotype control (3 citations) Goat anti-mouse IgG1-PE (2 citations) Mouse IgG1-PE (11 citations) Anti-IgG1-PE (2 citations)

8 protocols using igg1 pe

Multi-Color Flow Cytometry Immunophenotyping

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Antibody combination panels for four or five color immune-staining were as follows:

CD3PerCP, CD4 FITC, CD25APC, and rat Foxp3PE (all from eBioscience, San Diego, CA).

CD4 PercpCy5.5, HLA-DR APC, CD127 Pac Blue and rat Foxp3 PE (eBioscience, San Diego, CA).

CD4 PercpCy5.5, CCR5 FITC, CXCR4 APC (Biolegend, San Diego, CA) and Foxp3 PE.

CD4 PercpCy5.5, HIV-1 p24 FITC (Beckman Coulter, Brea, CA), Bcl-2 PE (BD Biosciences, San Jose, CA) and Foxp3 e-fluor660 (eBioscience).

CD4 PercpCy5.5, CD127 Pac Blue, Foxp3 PE, HIV-1 p24 FITC and IFN-γ Alexa Fluor 647 (Biolegend).

Isotype antibodies used included: IgG2a APC, IgG1 Pac Blue, rat IgG2a PE, IgG2a FITC, rat IgG2a AP, IgG1 FITC, IgG1 PE, IgG2a e-fluor660, and IgG1 Alexa Fluor 647.
The protocol and buffer set from eBioscience were used for all experiments involving intracellular staining. All samples were fixed and acquired within 1h of completion of staining, and analysis was by Miltenyi MACSQuant Flow cytometer.
Data were analyzed by FlowJo software (Treestar Inc, Ashland, OR) at the completion of study.

Hirsch C.S., Baseke J., Kafuluma J.L., Nserko M., Mayanja-Kizza H, & Toossi Z. (2016). Expansion and productive HIV-1 infection of Foxp3 positive CD4 T cells at pleural sites of HIV/TB co-infection. Journal of clinical & experimental immunology, 1(1), http://www.opastonline.com/wp-content/uploads/2016/11/expansion-and-productive-hiv-1-infection-of-foxp3-positive-cd4-t-cells-at-pleural-sites-of-hivtb-co-infection-jcei-16-007.pdf.

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Exosome Immunocapture and Flow Cytometry

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Immune capture and bead-based flow cytometry were carried out as described previously (8 (link),24 (link),25 (link)). In short, 10 µg exosomes in 100 µl PBS were incubated with 1 µg biotin-labeled anti-CD63 (Cat: 353018, Biolegend, San Diego, CA, USA) for 2 h at room temperature on a shaker. Next, 10 µl ExoCap Streptavidin magnetic beads (MBL Life Science, Woburn, MA, USA) were added and incubated for another 2 h at room temperature on a shaker. Samples were washed using a magnetic rack and subsequently stained with the following antibodies/isotype controls for 1 h at room temperature on a shaker: CD14-PE (0.5 µg, Cat: 12-0149-42) and IgG1-PE (0.5 µg, Cat: 12-4714-42) (both from eBioscience/Thermofisher Scientific), CD16-APC (0.8 µg, Cat: 36076) and IgG1-APC (0.8 µg, Cat: 400122) (both from Biolegend), CD44v3-APC (10 µl, FAB5088A) and IgG2b-APC (10 µl, IC0041A) (both from R&D, Minneapolis, MN, USA). The stained complexes were washed twice using a magnetic rack and finally resuspended in 300 µl PBS for flow cytometry. Detection was performed using a Gallios flow cytometer with Kaluza 1.0 software (Beckman Coulter, Brea, CA, USA) and 10000 events were acquired. Data are presented as relative fluorescent intensity (RFI) which is the mean fluorescence intensity of the stained sample divided by the mean fluorescence intensity of the corresponding isotype control.

Theodoraki M.N., Hofmann L., Huber D., Brunner C., Hoffmann T.K., Idel C., Fleckner J., Bruchhage K.L, & Pries R. (2023). Plasma‑derived CD16 exosomes and peripheral blood monocytes as correlating biomarkers in head and neck cancer. Oncology Letters, 25(5), 200.

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Macrophage Infection Quantification

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Macrophages were harvested after TrypLE^TM Express treatment (Gibco) and gentle scraping. Cells were fixed for 20 min in 4% paraformaldehyde (PFA), washed in PBS, and when stained, permeabilized for 30 min in PBS plus 0.2% BSA and 0.05% saponin. Infection was detected by GFP coding viruses and BST2 was detected using a coupled BST2-PE antibody (eBioscience) or IgG1-PE (eBioscience) as a control and analyzed on a FACS Verse (BD). For Vpu-Cherry expressing cells CYTOFLEX cytometer (Beckman Coulter) was used.

Gea-Mallorquí E., Zablocki-Thomas L., Maurin M., Jouve M., Rodrigues V., Ruffin N, & Benaroch P. (2020). HIV-2-Infected Macrophages Produce and Accumulate Poorly Infectious Viral Particles. Frontiers in Microbiology, 11, 1603.

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Flow Cytometry Analysis of Immune Cells

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Mouse anti-human CD3-PE-Cyanine7 (BioLegend, Clone: UCHT1), CD4-FITC (BioLegend, Clone: RPA-T4), CD8-PE (BioLegend, Clone: RPA-T8), CD25-APC (BioLegend, Clone: BC96), Foxp3-PE (eBioscience, Clone: 236A/E7), IgG1-PE (eBioscience, Clone: P3.6.2.8.1), IgG1-FITC (eBioscience, Clone: P3.6.2.8.1), IgG2b-PE-Cyanine7 (BD Pharmingen, Clone: A95-1) and IgG2a-APC (eBiocience, Clone: eBR2a) antibodies were used for flow cytometry analysis. Patients’ blood samples as well as peripheral blood and single-cell suspension of target organs of mice at the time of necropsy were analyzed by flow cytometry. Briefly, red blood cells were treated with Lysing Buffer (BD Pharm LyseTM) at 4°C for 15 min according to the protocol. Intracellular Foxp3 staining was performed according to the protocol (Fixation/Permeabilization Solution Kit; eBioscience). All samples were incubated with antibodies or isotype matched control IgG for 30 min in the dark. Then, flow cytometric analyses were performed on the Flow Cytometer (BD FACS Canto II) and further analyzed using FlowJo 10.0.

Bu X., Pan W., Wang J., Liu L., Yin Z., Jin H., Liu Q., Zheng L., Sun H., Gao Y, & Ping B. (2023). Therapeutic Effects of HLA-G5 Overexpressing hAMSCs on aGVHD After Allo-HSCT: Involving in the Gut Microbiota at the Intestinal Barrier. Journal of Inflammation Research, 16, 3669-3685.

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Immunophenotyping of AF-MSCs

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Cell surface markers of AF-MSCs were detected as described by Glemžaitė and Navakauskienė (14 (link)) using the following antibodies: CD44 (Invitrogen, Thermo Fisher Scientific, CA, USA), CD34 (Miltenyi Biotec, Bergisch Gladbach, Germany) and CD90 (Molecular Probes, Thermo Fisher Scientific, OR, USA) – FITC conjugated mouse anti-human, mouse anti-human CD105, labeled with PE (Invitrogen, Thermo Fisher Scientific, CA, USA) and mouse IgG2A-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), IgG1-FITC, IgG2b-FITC (Invitrogen, Thermo Fisher Scientific, CA, USA) or IgG1-PE (Molecular Probes, Thermo Fisher Scientific, OR, USA) as isotype controls. The expression of cell surface markers was measured using BD FACSCanto^TM II flow cytometer and BD FACSDIVA^TM software (BD Biosciences, CA, USA).

Gasiūnienė M., Petkus G., Matuzevičius D., Navakauskas D, & Navakauskienė R. (2019). Angiotensin II and TGF-β1 Induce Alterations in Human Amniotic Fluid-Derived Mesenchymal Stem Cells Leading to Cardiomyogenic Differentiation Initiation. International Journal of Stem Cells, 12(2), 251-264.

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Phenotypic Characterization of AF-MSCs

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For identification of the phenotype of AF-MSCs from passages 4-5, cells were collected by centrifugation at 1,200 rpm for 6 min, washed once in phosphate buffered saline (PBS) with 0.2% fetal calf serum (FCS), and centrifuged again. A total of 5 × 10⁵ cells were resuspended in 50 μL of PBS with 1% BSA and incubated with fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human antibodies against CD44 (Invitrogen), CD34 (Miltenyi Biotech), CD90 (Molecular Probes, Life technologies) or phycoerythrin- (PE-) labelled CD105 (Invitrogen), and appropriate isotype control mouse IgG2A-FITC (Miltenyi Biotec) or IgG1-PE (Molecular Probes, Life Technologies). Samples were incubated in the dark at 4°C for 30 min and analysis was performed on a flow cytometer BD FACSCanto II (Beckton and Dickinson) with BD FACSDiva software.

Savickiene J., Treigyte G., Baronaite S., Valiuliene G., Kaupinis A., Valius M., Arlauskiene A, & Navakauskiene R. (2015). Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis. Stem Cells International, 2015, 319238.

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Phenotypical Identification of AF-MSCs

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For phenotypical identification of AF-MSCs, at least 1 × 10⁵ cells for one assay were collected by centrifugation at 600 ×g for 5 min. Pelleted cells were washed twice in phosphate-buffered saline (PBS) supplemented with 0.2% foetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY, USA). Then cells were suspended in 50 μL PBS with 1% bovine serum albumin (BSA) and incubated with the following antibodies against cell surface markers: FITC conjugated mouse anti-human CD45 (BD Pharmingen, San Jose, CA, USA), CD34 (Miltenyi Biotec, Teterow, Germany), and CD90 (Molecular Probes, Life Technologies, Waltham, MA, USA) or PE labeled mouse anti-human CD105 (Invitrogen, Life Technologies, Waltham, MA, USA). Mouse IgG2A-FITC (Miltenyi Biotec, Teterow, Germany), IgG1-FITC (Invitrogen, Life Technologies, Waltham, MA, USA), or IgG1-PE (Molecular Probes, Life Technologies, Waltham, MA, USA) was used as isotype controls. Samples were incubated in the dark at 4°C for 30 min and washed twice with PBS with 1% BSA. Finally, cells were suspended in 200 μL PBS with 1% BSA and analyzed using Guava easyCyte 8HT flow cytometer (Millipore, USA) with InCyte 2.2.2 software.

Glemžaitė M, & Navakauskienė R. (2016). Osteogenic Differentiation of Human Amniotic Fluid Mesenchymal Stem Cells Is Determined by Epigenetic Changes. Stem Cells International, 2016, 6465307.

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Phenotyping AF-MSCs Using Flow Cytometry

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For identification of the phenotype of AF-MSCs from passages 4-5, cells were collected by centrifugation at 1,200 rpm for 6 min, washed once in phosphate buffered saline (PBS) with 0.2% fetal calf serum (FCS), and centrifuged again. A total of 5 × 10⁵ cells were resuspended in 50 μL of PBS with 1% BSA and incubated with fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human antibodies against CD44 (Invitrogen), CD34 (Miltenyi Biotech), CD90 (Molecular Probes, Life Technologies), or phycoerythrin- (PE-) labeled CD105 (Invitrogen) and appropriate isotype control—mouse IgG2A-FITC (Miltenyi Biotech) or IgG1-PE (Molecular Probes, Life Technologies). Samples were incubated in the dark at 4°C for 30 min and finally analyzed with the Millipore Guava® easyCyte 8HT flow cytometer, using the InCyte 2.2.2 software. Ten thousand events were collected for each sample.

Savickienė J., Baronaitė S., Zentelytė A., Treigytė G, & Navakauskienė R. (2016). Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy. Stem Cells International, 2016, 2019498.

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