Ebio1d3

Manufactured by Thermo Fisher Scientific

The EBio1D3 is a laboratory instrument designed for electrophoresis applications. It is capable of separating and analyzing biomolecules such as proteins and nucleic acids based on their size and charge.

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Lab products found in correlation

13 protocols using ebio1d3

Multiparametric T Cell Phenotyping

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Fc receptors were blocked with purified anti–mouse CD16/32 (2.4G2; BD). The cells were suspended in FACS buffer and stained at saturating conditions with the anti–mouse monoclonal antibodies against CD3 (145-2C11; eBioscience), CD4 (RM4-5; Invitrogen), CD44 (1M7; eBioscience), CD8 (53–6.7; eBioscience), PD-1 (RMP1-30; BioLegend), KLRG1 (2F1; BioLegend), Ly6C (HK1.4; eBioscience), CD62L (MEL-14; eBioscience), CD127 (A7R34; eBioscience), and CD43 (S7; BD). To exclude cells that may have nonspecifically bound to the tetramers, antibodies against non–T cell markers F4/80 (BM8; eBioscience), CD19 (eBio1D3; eBioscience), CD11c (N418; eBioscience), and CD11b (M1/70; eBioscience) were included in a dump channel. All surface staining was done at 4°C for 30 min, except staining for CXCR3 (CXCR3-173; eBioscience), CXCR5 (2G8; BD), and CCR7 (4B12; eBioscience), which was done at room temperature for 1 h. Samples were fixed in PBS containing 2% paraformaldehyde.

Moguche A.O., Shafiani S., Clemons C., Larson R.P., Dinh C., Higdon L.E., Cambier C.J., Sissons J.R., Gallegos A.M., Fink P.J, & Urdahl K.B. (2015). ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis. The Journal of Experimental Medicine, 212(5), 715-728.

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Multicolor Flow Cytometry Analysis of CAR T Cells

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10-color Gallios B43618 (Beckman Coulter, Indianapolis, IN) and 14-color Attune NxT (ThermoFisher Scientific) were used to acquire data. Analysis was performed with FlowJo software. Cells were counted with 123count eBeads (Thermo Fisher). Expression of CAR was detected by myc tag (9E10, Alexa Fluor 647, ThermoFisher) or violet-excitable GFP tag. DAPI (0.5 mg/ml, Sigma-Aldrich) or a LIVE/DEAD fixable yellow fluorescent dye (Thermo Fisher) were used to exclude dead cells in all experiments. Sorting of splenocytes after tissue processing was done using a BD FACSAria under sterile conditions. Antibodes to the following mouse proteins were used for flow cytometry: CD3ε (145-2C11,eBioscience, cat# 11-0031-85), CD3 (17A2, BioLegend, cat# 100233), CD4 (GK1.5, eBioscience, cat# 48-0041-80), CD4 (RM4-5, eBioscience, cat# 69-0042-80), CD8α (53-6.7, eBioscience, cat# 48-0041-80), CD19 (eBio1D3, eBioscience, cat# 48-0193-82), CD25 (PC61.5, eBioscience, cat# 25-0251-81), CD45 (30-F11, BioLegend, cat# 103106), CD80 (16-10A1, eBioscience, cat# 46-0801), CD86 (GL1, eBioscience, cat# 25-0862), CD223LAG-3 (C9B7W, eBioscience, cat# 12-5956-80), CD279 PD-1 (J43, eBiosceince, cat# 48-9985-82), and TIM3(8B.2C12, eBiosceince, cat# 48-5871-80).

Wijewarnasuriya D., Bebernitz C., Lopez A.V., Rafiq S, & Brentjens R. (2020). Excessive costimulation leads to dysfunction of adoptively transferred T cells. Cancer immunology research, 8(6), 732-742.

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Multiparametric Phenotyping of Liver Immune Cells

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Mouse cell surface molecule and intracellular IFN-γ and FoxP3 staining was performed as described (32 (link)). Liver NPC were treated with FcγR-blocking rat anti-mouse CD16/32 mAb (2.4G2) to prevent non-specific Ab binding. For cell surface staining, the NPC were incubated for 30 min with FITC-, phycoerythrin (PE)-, allophycocyanin (APC) -, Pacific Blue-, or PE-cyanin (Cy)7-conjugated mAbs to detect expression of CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11c (HL3), CD45 (30-F11), H2-K^b (AF6-88.5), NK1.1 (PK136) (BD Biosciences, San Diego, CA), CD19 (eBio 1D3, eBioscience, San Diego, CA) and F4/80 (BM8, BioLegend, San Diego, CA). For intracellular cytokine staining, cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% saponin, then stained with anti-mouse IFN-γ Ab (XMG1.2) (BioLegend). For Foxp3 staining, cells were fixed and permeabilized using Foxp3 Fix Permkit (eBioscience) and stained with anti-Foxp3 mAb (FJK-16s) (eBioscience). Appropriate Ig isotype controls were obtained from BD PharMingen (San Diego, CA). Flow cytometry was performed using a LSR Fortessa flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (version 7.6; TreeStar, Inc., Ashland, OR).

Yokota S., Yoshida O., Dou L., Spadaro A.V., Isse K., Ross M.A., Stolz D.B., Kimura S., Du Q., Demetris A.J., Thomson A.W, & Geller D.A. (2015). IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production. Journal of immunology (Baltimore, Md. : 1950), 194(12), 6045-6056.

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Flow Cytometry Cell Immunophenotyping

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Single cells suspensions were stained for 30 minutes in PBS (with 0.5% BSA and 2 mM EDTA) with ‘cocktails’ of the following antibodies CD3ε PECy7 (1:200) (145-2C11), CD19 APC-Cy7 (1:100) (eBio1D3) (from EBiosciences). Cells were then washed twice in PBS (with 0.5% BSA and 2 mM EDTA), resuspended in PBS (with 0.5% BSA and 2 mM EDTA) and then analyzed using a Cyan-ADP (Dako) with forward/side scatter gates set to exclude non-viable cells. Data were analyzed with FlowJo software (Tree Star, Oregon, USA).

Chimen M., McGettrick H.M., Apta B., Kuravi S.J., Yates C.M., Kennedy A., Odedra A., Alassiri M., Harrison M., Martin A., Barone F., Nayar S., Hitchcock J.R., Cunningham A.F., Raza K., Filer A., Copland D.A., Dick A.D., Robinson J., Kalia N., Walker L.S., Buckley C.D., Nash G.B., Narendran P, & Rainger G.E. (2015). Homeostatic regulation of T cell trafficking by a B cell derived peptide is impaired in autoimmune and chronic inflammatory disease. Nature medicine, 21(5), 467-475.

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Adoptive T and B cell transfer in TAC murine model

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Wild-type B and T cells were isolated from 10–12-week-old male C57BL6/J male mice, respectively, with B Cell Isolation Kit and Pan T Cell Isolation Kit II (Miltenyi Biotec) on an AutoMACS. Purity was assessed by staining with anti-mouse CD3ɛ (145-2C11, BioLegend) or anti-mouse CD19 (eBio1D3, eBioscience), and analysed by flow cytometry. C57BL6/J Il10 KO male mice, before basal echocardiography screening, were injected intravenously with 2 × 10⁶ WT T or B cells. Mice underwent TAC surgery and were injected with abatacept starting on day 2 after surgery.

Kallikourdis M., Martini E., Carullo P., Sardi C., Roselli G., Greco C.M., Vignali D., Riva F., Ormbostad Berre A.M., Stølen T.O., Fumero A., Faggian G., Di Pasquale E., Elia L., Rumio C., Catalucci D., Papait R, & Condorelli G. (2017). T cell costimulation blockade blunts pressure overload-induced heart failure. Nature Communications, 8, 14680.

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Multiparameter Flow Cytometry Analysis

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Antibodies against CD3e (145-2C11), CD4 (GK1.5), NK1.1 (PK136), CD19 (eBio1D3), CD24 (30-F1), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD48 (HM48-1), CXCR5 (SPRCL5), GL7 (GL-7), GATA3 (TWAJ), IgD (11-26C), ICOS (7E.17G9), Ly9 (Ly9ab3), PD1 (J43), PLZF (Mags.21F7), RORγt (AFKJS-9), 2B4 (eBio244F4), 7-AAD, and streptavidin were purchased from eBioscience. Antibodies against CD138 (281–2) and Fas (Jo2) were purchased from BD. Antibodies against SLAM (12F12), CD84 (CD84.7), and Ly-108 (330-AJ) were purchased from BioLegend, whereas CRACC antibody (clone 520914) was provided by R&D Systems. Antibodies against Egr2 were purchased from Abcam, and goat anti–rabbit IgG (H+L) was purchased from Thermo Fisher Scientific. CD1d-tetramer (19156, loaded with PBS57) and empty CD1d control were kindly provided by the National Institutes of Health Tetramer Core Facility.

Chen S., Cai C., Li Z., Liu G., Wang Y., Blonska M., Li D., Du J., Lin X., Yang M, & Dong Z. (2017). Dissection of SAP-dependent and SAP-independent SLAM family signaling in NKT cell development and humoral immunity. The Journal of Experimental Medicine, 214(2), 475-489.

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Antibody Protocols for Neurodegenerative Markers

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Antibodies to poly‐GA (1:500, Proteintech, 24492‐1‐AP), GFP (1:1,000, Clontech, 632592), Iba1 (1:500, Wako, 091‐19741), TDP‐43 (1:500, Proteintech, 10782‐2‐AP), calnexin (1:3,000, Enzo Life Science, SPA‐860F), CD3 (eBioscience, 17A2), anti‐CD4 (clone: RM4‐5, eBioscience), CD8 (eBioscience, 53‐6.7), CD19 (eBioscience, eBio1D3), CD45 (eBioscience, 30‐F11), and CD11b (eBioscience, M1/70) are commercially available. In addition to our previous anti‐GA clone 5F2 (purified mouse monoclonal, biotinylated 1:1,000, Sulfo‐Tagged 10 ng/μl) used for immunoassay, we used clone 1A12 established from the same immunization (Mackenzie et al, 2013), which shows superior staining specificity (mouse monoclonal, IgG1, WB 1:50, IHC 1:100).

Zhou Q., Mareljic N., Michaelsen M., Parhizkar S., Heindl S., Nuscher B., Farny D., Czuppa M., Schludi C., Graf A., Krebs S., Blum H., Feederle R., Roth S., Haass C., Arzberger T., Liesz A, & Edbauer D. (2019). Active poly‐GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model. EMBO Molecular Medicine, 12(2), e10919.

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Immunomagnetic Cell Separation Protocol

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Splenocyte preparations were made and counted as above. Cells were washed twice with 10-ml MACS buffer (PBS/5% BSA/2 mM EDTA) and then resuspended in MACS buffer (1 ml/10⁸ cells) for staining using antibodies to CD19 (eBio1D3; eBioscience), Thy1.2 (30-H12; eBioscience) and Ter119 (TER-119; eBioscience). Cells were incubated with antibody for 25 min. before washing twice with MACS buffer. Cells were resuspended in 1-ml MACS buffer containing MACS® anti-biotin microbeads (13 μl/10⁸ cells; Miltenyi Biotec, North Ryde, NSW, Australia) and incubated for 30 min. on ice. Cells were then washed, resuspended in 500 μl MACS buffer and added on to pre-washed MACS® LS columns, which were washed three times with 3-ml MACS buffer. Flow-through containing cells was collected, cells were washed and checked for depletion by antibody staining and flow cytometry.

Ji J., Griffiths K.L., Milburn P.J., Hirst T.R, & O’Neill H.C. (2015). The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells. Journal of Cellular and Molecular Medicine, 19(8), 2019-2031.

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Flow Cytometry Cell Immunophenotyping

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Isolation and Culture of Intestinal ILC2

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Cells were purified by flow cytometric sorting from naïve C57BL/6J, Pdcd1^–/– or Csf2^–/– mice (Supplementary Fig. 9 and Extended Data Fig. 10). Lamina propria single cell suspensions were stained with antibodies for anti-CD45 (30-F11, BD Biosciences, 1/1,200 dilution), anti-CD3ε (145-2C11, eBioscience, or 17A2, BioLegend, 1/200 dilution), anti-CD19 (1D3, BD Biosciences, or eBio1D3, eBioscience, 1/800 dilution), anti-TCRβ (H57-597, eBioscience, 1/200 dilution), anti-CD11b (M1/70, BD Biosciences, 1/800 dilution), anti-c-kit (2B8, eBioscience, 1/300 dilution), anti-NK1.1 (PK136, BD Biosciences, 1/400 dilution), anti-Sca-1 (D7, BD Biosciences, 1/300 dilution) and anti-KLRG1 (2F1, BD Biosciences, 1/200 dilution) antibodies. Intestinal ILC2 were identified as: CD45⁺CD3^-CD19^-TCRβ^-CD11b^-NK1.1^-c-kit^-Sca-1⁺KLRG1^+/- or CD45⁺CD3^-CD19^-TCRβCD11b^-NK1.1^-c-kit^-CD90.2⁺CD127⁺KLRG1⁺. 2.5-8.5×10³ purified cells were cultured in 96 well plates with 40ng/ml rhIL-7 and rmIL-33 (both from PeproTech) in complete media. After two to five days of culture, cells were collected and stained to assess surface and intracellular cytokine expression. The ILC2-conditioned media was collected to supplement eosinophil cultures at a 1:1 ratio (Supplementary Fig. 9).

Jacquelot N., Seillet C., Wang M., Pizzolla A., Liao Y., Hediyeh-zadeh S., Grisaru-Tal S., Louis C., Huang Q., Schreuder J., Souza-Fonseca-Guimaraes F., de Graaf C.A., Thia K., Macdonald S., Camilleri M., Luong K., Zhang S., Chopin M., Molden-Hauer T., Nutt S.L., Umansky V., Ciric B., Groom J.R., Foster P.S., Hansbro P.M., McKenzie A.N., Gray D.H., Behren A., Cebon J., Vivier E., Wicks I.P., Trapani J.A., Munitz A., Davis M.J., Shi W., Neeson P.J, & Belz G.T. (2021). Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent anti-tumor immunity in melanoma. Nature immunology, 22(7), 851-864.

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