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Anti 1 a 1 e

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Anti-I-A/I-E is a lab equipment product that detects the mouse major histocompatibility complex (MHC) class II antigens I-A and I-E. It is used for flow cytometric analysis and cell sorting applications.

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Lab products found in correlation

Propidium iodide, by Thermo Fisher Scientific (6 mentions) Anti cd45, by BioLegend (5 mentions) Anti cd11c, by BioLegend (4 mentions) Anti cd4, by BioLegend (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Anti cd11b, by BioLegend (3 mentions) Anti ifn γ, by BioLegend (3 mentions) Anti cd3, by BioLegend (3 mentions) Anti nk1, by BioLegend (2 mentions) Anti il 17a, by BioLegend (2 mentions) Anti cd69, by BioLegend (2 mentions) Anti foxp3 fjk 16s, by Thermo Fisher Scientific (2 mentions) Anti b220 cd45ro, by BioLegend (2 mentions) Fluorochrome conjugated anti lyve1 aly7, by Thermo Fisher Scientific (2 mentions) Anti s1pr1 clone 713412, by R&D Systems (2 mentions) Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (2 mentions) Anti cd8, by BioLegend (2 mentions) Anti cd44, by BioLegend (2 mentions) Anti ly6c, by BioLegend (2 mentions) Anti cd31, by BioLegend (2 mentions)

Close spelling variants of this product

Anti-I-A/I-E (M5/114.15.2, (5 citations) Anti-I-A/I-E-BV605 (4 citations) PE anti-mouse I-A/I-E (4 citations) Rat anti-I-A/I-E (4 citations) Anti-I-A/I-E Pacific Blue (3 citations) Anti-mouse I-A/I-E (3 citations) Anti-mouse I-A/I-E antibody (3 citations) Anti-mouse I-A/I-E-BV605 (3 citations) Anti-I-A/I-E-PerCP (clone M5/114.15.2, (3 citations) APC anti-mouse I-A/I-E antibody (2 citations)

9 protocols using anti 1 a 1 e

1

Multiparametric Flow Cytometry for Immune Cell Analysis

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To determine chimerism, splenocytes were stained with anti‐H‐2Dd‐FITC (clone: 34–2–12) and anti‐H‐2Db‐PE (clone: KH95) (all from Biolegend). Treg cells were determined in the spleens according to manufacturer's instructions using a kit from eBioscience (San Diego, CA). Spleen cells were also characterized using anti‐CD11b (clone: M1/70), anti‐CD11c (clone: N418), anti‐B220/CD45R (clone: RA3‐6B2), anti‐I‐A/I‐E (clone: M5/114.152), anti‐CD4 (clone: RM4‐5 and GK1.5), anti‐pDCA1 (clone: 927), anti‐CD8α (clone: 100712), anti‐TCR‐β (clone: H57‐597), all from Biolegend, and a viability marker (LIVE/DEAD® Fixable Near‐IR Dead Cell Stain Kit, Life Technologies, Eugene, OR). For MACS cell purification, we used anti‐CD11c (cat. 130‐052‐001), anti‐CD4 (cat. 130‐049‐201), and anti‐CD8 (cat. 130‐049‐301) microbeads (Miltenyi Biotec Bergisch Glabach, Germany). For the in vitro functional assays, DC subpopulations were purified from spleens by MACS. CD11c cells were then sorted by FACS (BD FacsAria III) using APC/Cy7‐labeled anti‐B220, PercP‐labeled anti‐I‐A/I‐E, APC‐labeled anti‐pDCA1, PE‐labeled anti‐CD11b and PE/Cy7‐CD8a. We obtained 90.9 ± 1.35% purity for CD8αcDCs, 95.1 ± 1% for CD11b cDCs and 94.± 0.3% for pDCs. All cells were acquired using a FACS‐LSRFortessa according to BD bioscience protocols and analyzed by FlowJo software version 9.8.1.
Gaignage M., Marillier R.G., Uyttenhove C., Dauguet N., Saxena A., Ryffel B., Michiels T., Coutelier J, & Van Snick J. (2017). Mouse nidovirus LDV infection alleviates graft versus host disease and induces type I IFN‐dependent inhibition of dendritic cells and allo‐responsive T cells. Immunity, Inflammation and Disease, 5(2), 200-213.
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2

Multiparametric Flow Cytometry Analysis of Immune Cells

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Cells were incubated with antibodies 4 °C 20 min. Cells were examined by Aria II Flow Cytometer (BD Bioscience, USA). Cells were gated as follows: DCs (F4/80− CD11c+ IA/IE+), macrophages (CD11b+ F4/80+) and M1 macrophages (CD11b+ F4/80+ CD206− MHC IIhigh), M2 macrophages (CD11b+ F4/80+ CD206+ MHC IIlow). Intracellular cytokine staining: 2 µL mL−1 Cell Activation Cocktail (with Brefeldin A) (Cat: 423,303, Biolegend, USA) was used to incubate cells at 37 °C in a CO2 incubator for 6 h. Then, the Fixation/Permeabilization Solution Kit (Cat: 554,714, BD Biosciences, USA) was used to stimulus cells. After cell fixation and permeabilization (fixation/permeabilization solution, 100 uL/106 cells, 4 °C, 30 min), the BD Perm/Wash™ Buffer is used to wash the cells and to dilute the IFN-γ antibody for staining (4 °C, 30 min). Antibodies: The following were purchased from BioLegend: anti-IA/IE (1:3200, Clone: M5/114.15.2, Cat: 107,630), anti-CD206 (1:200, Clone: C068C2, Cat: 141,705), anti-Ki67 (1:200, Clone: 16A8, Cat: 652,403), anti-IFN-γ (1:100, Clone: XMG1.2, Cat: 505,805). The following were purchased from eBioscience: anti-CD11b (1:200, Clone: M1/70, Cat: 47–0112-82), anti-CD11c (1:200, Clone: N418, Cat: 45–0114-82), anti-F4/80 (1:200, Clone: BM8, Cat: 17–4801-82).
Zhao Q., Shi M., Yin C., Zhao Z., Zhang J., Wang J., Shen K., Zhang L., Tang H., Xiao Y, & Zhang Y. (2020). Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis. Nano-Micro Letters, 13, 28.
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3

Multiparametric Flow Cytometry and Microscopy

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The following fluorochrome-conjugated antibodies and reagents were used: anti-CD3 (145–2C11, 5 μg/ml for flow cytometry and 10μg/ml for microscopy), anti-CD4 (RMA 4–5, 1 μg/ml), anti-CD8 (53–6.7, 2.5 μg/ml), anti-CD44 (IM7, 2.5μg/ml), anti-B220/CD45RO (RA3–6B2, 2.5 μg/ml), anti-CD69 (H1.2F3, 2μg/ml), anti-NK1.1 (PK136, 0.5 mg/ml), anti-I-A/I-E (M5/114.15.2, 0.2 mg/ml), anti-CD11c (N418, 0.2 mg/ml), anti-CD14 (Sa14–2, 0.2 mg/ml), anti-CD45.1 (A20, 0.5 mg/ml), anti-CD45.2 (104, 0.5 mg/ml), anti-IL-17A (TC11–18H10.1, 0.2 mg/ml), anti-IFN-γ (XMG1.2, 0.2 mg/ml), streptavidin (2.5 μg/ml for flow cytometry and 1.25μg/ml for microscopy) (all from BioLegend). Fluorochrome-conjugated anti-Lyve1 (ALY7, 2.5 μg/ml), anti-FoxP3 (FJK-16 s, 0.5 mg/ml) were from eBioscience. Anti-S1PR1 (clone 713412) was from R&D Systems. CD4 (RMA 4–5, 0.2 mg/ml) was from BD Biosciences. Propidium iodide (0.75μg/ml), DAPI (0.125μg/ml) or LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher L23105) was used to exclude dead cells.
Okuniewska M., Fang V., Baeyens A., Raghavan V., Lee J.Y., Littman D.R, & Schwab S.R. (2021). SPNS2 enables T cell egress from lymph nodes during an immune response. Cell reports, 36(2), 109368.
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4

Isolation and Characterization of Mouse Pancreatic Immune Cells

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Mouse pancreatic tissues were digested by digestion buffer (1 mg/ml collagenase IV [Sigma-Aldrich] and 10 U/ml DNase I [Sigma-Aldrich] in DMEM) at 37 °C for 30 min. The digested tissues were pushed through a 70-µm cell strainer and washed by 40 ml of DMEM without FBS. The digested cells were resuspended in 37% Percoll, with the same volume of 70% Percoll on the bottom of the tube. After centrifugation at 800 g for 20 min with break off, the immune cells were isolated from the 37%/70% interface and rinsed by PBS. The cells were first stained by Zombie UV (BioLegend, #423107, 1:500), then were incubated with a cocktail comprising the following antibodies in cell staining buffer (#420201, BioLegend): anti-CD45 (#103131, 1:150), anti-CD3ε (#100355, 1:60), anti-B220 (#103231, 1:200), anti-Gr1 (#108438, 1:150), anti-CD11b (#101215, 1:250), anti-F4/80 (#123131, 1:400), anti-Ly6C (#128033, 1:40), anti-Ly6G (#127651, 1:80), anti-CD4 (#100405, 1:200), anti-IA/IE (#107607, 1:500, all from BioLegend), and anti-CD8a (#564297, 1:200, BD Biosciences). After washing, the stained cells were submitted for multiple-color flow cytometry analysis. The data were collected on LSR Fortessa X-20 analyzer with FACSDiva software version 8.0 (BD) and analyzed using FlowJo version 10 (BD).
Liu Y., Deguchi Y., Wei D., Liu F., Moussalli M.J., Deguchi E., Li D., Wang H., Valentin L.A., Colby J.K., Wang J., Zheng X., Ying H., Gagea M., Ji B., Shi J., Yao J.C., Zuo X, & Shureiqi I. (2022). Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ. Nature Communications, 13, 2665.
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5

Multiparametric Flow Cytometry and Microscopy

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The following fluorochrome-conjugated antibodies and reagents were used: anti-CD3 (145–2C11, 5 μg/ml for flow cytometry and 10μg/ml for microscopy), anti-CD4 (RMA 4–5, 1 μg/ml), anti-CD8 (53–6.7, 2.5 μg/ml), anti-CD44 (IM7, 2.5μg/ml), anti-B220/CD45RO (RA3–6B2, 2.5 μg/ml), anti-CD69 (H1.2F3, 2μg/ml), anti-NK1.1 (PK136, 0.5 mg/ml), anti-I-A/I-E (M5/114.15.2, 0.2 mg/ml), anti-CD11c (N418, 0.2 mg/ml), anti-CD14 (Sa14–2, 0.2 mg/ml), anti-CD45.1 (A20, 0.5 mg/ml), anti-CD45.2 (104, 0.5 mg/ml), anti-IL-17A (TC11–18H10.1, 0.2 mg/ml), anti-IFN-γ (XMG1.2, 0.2 mg/ml), streptavidin (2.5 μg/ml for flow cytometry and 1.25μg/ml for microscopy) (all from BioLegend). Fluorochrome-conjugated anti-Lyve1 (ALY7, 2.5 μg/ml), anti-FoxP3 (FJK-16 s, 0.5 mg/ml) were from eBioscience. Anti-S1PR1 (clone 713412) was from R&D Systems. CD4 (RMA 4–5, 0.2 mg/ml) was from BD Biosciences. Propidium iodide (0.75μg/ml), DAPI (0.125μg/ml) or LIVE/DEAD Fixable Blue Dead Cell Stain (ThermoFisher L23105) was used to exclude dead cells.
Okuniewska M., Fang V., Baeyens A., Raghavan V., Lee J.Y., Littman D.R, & Schwab S.R. (2021). SPNS2 enables T cell egress from lymph nodes during an immune response. Cell reports, 36(2), 109368.
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6

Isolation and Characterization of Microglia

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Microglia cells were isolated through Percoll gradient or Miltenyi Adult Brain Dissociation kit, as described in RNA‐Seq Analysis. Microglia staining occurred in 1X dPBS−/− (Thermo Fischer) supplemented with 0.5% FBS, 0.4% 0.5 M EDTA, and 1% HEPES at 4°C for 20 min. Cells were stained with the following antibodies: anti‐CD11b (clone:M1/70, eBioscience), anti‐CD45 (clone:30‐F11, eBioscience), anti‐F480 (clone:BM8, eBiosience), anti‐CX3CR1 (clone:SA011F11, Biolegend), anti‐CD31 (clone:MEC13.3, Biolegend), anti‐CCR3 (clone:J073E5, Biolegend), anti‐ I‐A/I‐E (clone:M5/114.15.2, Biolegend), anti‐CD80 (clone:16‐10A1, eBioscience), anti‐CD86 (clone:GL1, eBioscience), and anti‐TLR4 (clone:SA15‐21, Biolegend). Following staining, cells were washed twice and fixed in a 1:1 solution of supplemented dPBS−/−: 4% paraformaldehyde overnight at 4°C. After fixation, cells were resuspended in supplemented dPBS−/−and enumerated via flow cytometry (BD LSR II with 561 laser). Microglia populations were identified as CD11bhigh/CD45low. Subsequent data was analyzed using FlowJo software (v. 10.5.3).
Bauer K.C., York E.M., Cirstea M.S., Radisavljevic N., Petersen C., Huus K.E., Brown E.M., Bozorgmehr T., Berdún R., Bernier L., Lee A.H., Woodward S.E., Krekhno Z., Han J., Hancock R.E., Ayala V., MacVicar B.A, & Finlay B.B. (2022). Gut microbes shape microglia and cognitive function during malnutrition. Glia, 70(5), 820-841.
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7

Spike-specific T cell responses post-booster

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Five weeks after booster, mouse splenocytes were isolated and incubated for 6 h at 37°C with BD GolgiPlug (BD Bioscience 555028) and with or without spike peptide pools (JPT Peptide Technologies PM-WCPV-S-1). Cells were washed, stained, and analyzed as described in the section “T follicular helper and B cell phenotype detection in lymph node by flow cytometer.” Antibodies for extracellular antigens are CD3 (BioLegend 100220), CD4 (BioLegend 100540), CD8 (BD Bioscience 557654), CD44 (BioLegend 103040), anti-I-A/I-E (BioLegend 107608), and live/dead fixable yellow dead cell staining (Thermo Scientific L34968), and for intracellular antigens are TNF-α (BioLegend 506304) and IFN-γ (BioLegend 505810).
Hu Q., Zhao Y., Shaabani N., Lyu X., Powers C., Sun H., Cruz V., Stegman K., Xu J., Fossier A., Huang Y., Ho G., Kao Y., Wang Z., Wang Z., Hu Y., Zheng Y., Kyaw L., Zuluaga C., Wang H., Pei H., Allen R., Xie H., Ji H, & Chen R. (2022). Chimeric mRNA-based COVID-19 vaccine induces protective immunity against Omicron and Delta variants. Molecular Therapy. Nucleic Acids, 30, 465-476.
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8

Tumor Immune Cell Profiling

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Tumor single-cell suspensions were resuspended in flow cytometry buffer (PBS, 2% FBS, and 2 mmol/L EDTA), blocked with Fc block, and then stained with different fluorochrome-conjugated antibodies for 15 min on ice. The following antibodies were used at the noted diluitions: anti-CD31 (Biolegend 102506, 1:200), anti-αSMA (Sigma C6198, 1:100), anti-CD8 (eBioscience 25-0081-82, 1:100), anti-FoxP3 (eBioscience 61-5773-82, 1:50), anti-CD11b (Biolegend 101228, 1:100), anti-PDPN (Biolegend 127410, 1:100), anti-CD4 (BD 563790, 1:100), anti-CD45 (BD 564279, 1:600), anti-CD45.2 (BD 564880, 1:400), anti-CD90 (Biolegend 140317, 1:100), anti-CD3 (Biolegend 100229, 1:100), anti-CD26 (eBioscience 45-0261-82, 1:100), anti-Ly6c (Biolegend 128026, 1:100), anti-I-A/I-E (Biolegend 107632, 1:100), and anti-CD73 (Biolegend 127217, 1:100). Samples were run on a BD flow cytometry Fortessa instrument and analyzed with FlowJo.
Grauel A.L., Nguyen B., Ruddy D., Laszewski T., Schwartz S., Chang J., Chen J., Piquet M., Pelletier M., Yan Z., Kirkpatrick N.D., Wu J., deWeck A., Riester M., Hims M., Geyer F.C., Wagner J., MacIsaac K., Deeds J., Diwanji R., Jayaraman P., Yu Y., Simmons Q., Weng S., Raza A., Minie B., Dostalek M., Chikkegowda P., Ruda V., Iartchouk O., Chen N., Thierry R., Zhou J., Pruteanu-Malinici I., Fabre C., Engelman J.A., Dranoff G, & Cremasco V. (2020). TGFβ-blockade uncovers stromal plasticity in tumors by revealing the existence of a subset of interferon-licensed fibroblasts. Nature Communications, 11, 6315.
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Comprehensive Immune Profiling of Tumor Samples

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Tumors were collected, minced, and digested with collagenase B (0.5 mg/mL, Roche) and hyaluronidase (0.5 mg/mL, Absin) at 37°C for 1 hour. Suspensions were filtered through 40 µm cell strainers. Then, cells were suspended and stained with Fixable Viability Stain 700 (BD Biosciences). Cells were stained according to the standard protocol for flow cytometry. The following antibodies and buffers were used (all reagents from BD Biosciences unless otherwise indicated): anti-CD45 (560510), anti-CD3e (562600), anti-CD8α (563068), anti-CD25 (553075), anti-CD69 (566500), anti-Ki67 (556027), anti-TNF-α (563943), anti-IFN-γ (560660), anti-Perforin (ThermoFisher, 11-9392-82), anti-Granzyme-B (BioLegend, 372204), anti-CD11c (566504), anti-I-A/I-E (BioLegend, 107608), anti-CD80 (560016), anti-CD86 (561962), Brilliant Stain Buffer (563794), and FOXP3/Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00). Cells per 100 mg tumor were counted by Beckman Vi-Cell Auto. Flow cytometry was performed with Beckman CytoFLEX LX.
Yi M., Wu Y., Niu M., Zhu S., Zhang J., Yan Y., Zhou P., Dai Z, & Wu K. (2022). Anti-TGF-β/PD-L1 bispecific antibody promotes T cell infiltration and exhibits enhanced antitumor activity in triple-negative breast cancer. Journal for Immunotherapy of Cancer, 10(12), e005543.
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