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Mhcii m5 114.15.2

Manufactured by Thermo Fisher Scientific
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The MHCII (M5/114.15.2) is a lab equipment product manufactured by Thermo Fisher Scientific. It functions as an antibody that recognizes major histocompatibility complex class II (MHC II) molecules. The core purpose of this product is to facilitate the identification and analysis of MHC II-expressing cells.

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Lab products found in correlation

F4 80 bm8, by Thermo Fisher Scientific (3 mentions) Facscanto 2, by BD (3 mentions) Fixable viability dye, by Thermo Fisher Scientific (3 mentions) Cd3 17a2, by BioLegend (2 mentions) Cd4 gk1, by BioLegend (2 mentions) B220 ra3 6b2, by Thermo Fisher Scientific (2 mentions) Cd11b m1 70, by BioLegend (2 mentions) Ly6c hk1, by BioLegend (2 mentions) Dnase 1, by Roche (2 mentions) Cd64 x54 5 7, by BioLegend (2 mentions) Cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Ly6g 1a8, by BioLegend (1 mentions) Cd8 53 6, by Thermo Fisher Scientific (1 mentions) Cd69 h1.2f3, by BioLegend (1 mentions) Collagenase type ia, by Merck Group (1 mentions) Cd44 im7, by BioLegend (1 mentions) Granzyme b ngzb, by Thermo Fisher Scientific (1 mentions) Tcrβ h57 597, by BioLegend (1 mentions) Nk1.1 pk136, by BioLegend (1 mentions) Cd45 30 f11, by BioLegend (1 mentions)

14 protocols using mhcii m5 114.15.2

1

Multiparameter Flow Cytometry of Tumor Cells

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Tumor tissues were digested both mechanically by chopping with razor blades and chemically with 1mg/mL type IA collagenase (Sigma-Aldrich) for 30 minutes at 37°C. Following digestion, cell suspensions were washed, filtered and stained as previously described (O’Sullivan et al., 2012 (link)). The following antibodies were used: Ly6C (ER-MP20, Serotec), MHCII (M5/114 15.2, eBioscience), Ly6G (1A8, Biolegend), CD8 (53-6.7, eBioscience), CD44 (IM7, Biolegend), CD3 (17A.2, Biolegend), CD4 (GK1.5, Biolegend), CD69 (H1.2F3, Biolegend), Granzyme B (NGZB, eBioscience), IFNγ (XMG 1.2, Biolegend), TCRβ (H57-597, Biolegend), B220 (RA3-6B2, eBioscience), NK1.1 (PK136, Biolegend), CD11b (M1/70, eBioscience), CD45 (30-F11, Biolegend). Stained cell suspensions were analyzed on a BD FACS CANTO II (BD Biosciences).
Saddawi-Konefka R., Seelige R., Gross E.T., Levy E., Searles S.C., Washington A J.r., Santosa E.K., Liu B., O’Sullivan T.E., Harismendy O, & Bui J.D. (2016). Nrf2 induces IL-17D to mediate tumor and virus surveillance. Cell reports, 16(9), 2348-2358.
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2

Quantifying Peritoneal Macrophage Subsets

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Two methods were used to quantify peritoneal macrophages. Total peritoneal cells were counted in a hemacytometer or using an automated cell counter. Then this number was multiplied by the percent of macrophages stained for CD115 (AFS98; eBioscience), ICAM-2 (3C4; BioLegend) and F4/80 (BM8, eBioscience) by flow cytometry. These antibodies allow us to distinguish the minor and major peritoneal macrophage populations (Gautier et al., 2012 (link)), with only the major macrophage population expressing ICAM-2 (Gautier et al., 2012 (link)). Alternatively, peritoneal cells obtained by lavage were incubated with FITC-conjugated anti–ICAM-2 mAb, and then a manual count of the fraction of ICAM-2+ cells was made and multiplied by the total number of peritoneal cells. Both methods yielded similar results. Other reagents and mAbs used in flow cytometry were Annexin V (Miltenyi Biotec) and antibodies against Ki67 (B56; BD), active caspase 3 (C92-605; BD), pH3 (D2C8; Cell Signaling technology), TIMD4 (RMT4-54; eBioscience), CD206 (MR5D3; Serotec), LYVE-1 (Abcam), Siglec F (E50-2440; BD), CD11b (M1/70; eBioscience), CD5 (53–7.3; BD), B220 (RA3-6B2; eBioscience), ly 6C/G (RB6-8C5; eBioscience), Ly6-G (1A8; BD), TCRb (H57-597; eBioscience), MHC-II (M5/114.15.2; eBioscience), and MARCO (ED31; Serotec).
Gautier E.L., Ivanov S., Williams J.W., Huang S.C., Marcelin G., Fairfax K., Wang P.L., Francis J.S., Leone P., Wilson D.B., Artyomov M.N., Pearce E.J, & Randolph G.J. (2014). Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. The Journal of Experimental Medicine, 211(8), 1525-1531.
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3

Comprehensive Immune Cell Profiling by Flow Cytometry

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Samples were blocked with 2% normal mouse serum or mouse Fc block (2.4G2, BD Biosciences). Fixable yellow (Invitrogen, L34959) was used to stain live/dead cells. Anti-mouse antibodies used were CD45.2 (104, Tonbo Biosciences), CD3 (500A2, BD Bioscience), TCRβ (H57–597, eBioscience), CD8 (53-6.7, BioLegend), CD4 (GK1.5, BioLegend), FoxP3 (FJK-16S, eBioscience), Ly6C (HK1–4, BioLegend), CD11b (M1/70, BioLegend), F4/80 (BM8, eBioscience), MHC II (M5/114.15.2, eBioscience), CD11c (N418, BioLegend), NK1.1 (PK136, BD Biosciences), IFNγ (XMG1.2, Tonbo Biosciences), H-2Db (28-14-8, eBioscience), H-2Kb (AF6–88.5.5.3, eBioscience), PD-1 (29 F.1A12, BioLegend), and PD-L1 (MIH5, eBioscience). Fluorescence was measured on BD LSR FortessaTM X-20 or BD FACSymphonyTM flow cytometer (BD Biosciences, North Ryde, New South Wales, Australia) and data analysed using FlowJo, LLC software.
Lelliott E.J., Cullinane C., Martin C.A., Walker R., Ramsbottom K.M., Souza-Fonseca-Guimaraes F., Abuhammad S., Michie J., Kirby L., Young R.J., Slater A., Lau P., Meeth K., Oliaro J., Haynes N., McArthur G.A, & Sheppard K.E. (2019). A novel immunogenic mouse model of melanoma for the preclinical assessment of combination targeted and immune-based therapy. Scientific Reports, 9, 1225.
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4

Comprehensive B-cell Immunophenotyping by Flow Cytometry

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Erythrocyte‐lysed single‐cell suspensions were blocked with anti‐CD16/32 for 15 min and stained with appropriate antibodies for 30 min on ice. The following stains and antibodies were used for immunophenotyping: B220 (RA3‐6B2), CD25 (M‐A251), CD69 (H1.3F2), CD86 (GL1), and CD95 (Jo2) from BD Bioscience; CD19 (1D3), CD23 (B3B4), CD38 (90), CD93 (AA4.1), IgM (II/41), and MHCII (M5/114.15.2) from eBioscience; and CD21/CD35 (7E9), CD45.1 (A20), CD138 (281‐2), and c‐kit (2B8) from BioLegend.
In spleen, follicular and marginal zone cells were detected based on CD23 and CD21/35 expression in the mature cell population (B220+CD93−). Transitional populations were detected in the immature population (B220+CD93+) based on expression of CD23 and IgM. Germinal center cells were defined as B220+ GL7+Fas+CD38‐ population; plasma cells were detected by expression of CD138. In the bone marrow, early B‐cell populations were defined as follows: pre‐pro‐B (B200+c‐kit+CD25−CD19−), pro‐B (B200+c‐kit+CD25‐CD19+), and pre‐B cells (B200+c‐kit‐CD25+CD19+).
Malinova D., Wasim L., Newman R., Martínez‐Riaño A., Engels N, & Tolar P. (2021). Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses. EMBO Reports, 22(9), e51328.
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5

Multiparameter Flow Cytometry Analysis

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For surface markers, single-cell suspensions were stained with relevant fluorochrome-conjugated monoclonal antibodies(mAbs): anti-mouse CD40 (HM40-3), CD80 (16-10A1), CD86 (GL1), and MHCII (M5/114.15.2) from eBioscience, anti-mouse CD11b (M1/70), Gr-1 (RB6-8C5), Ly6G (1A8), and Ly6C (HK1.4) from Biolegend, For intracellular staining, cells were stimulated with PMA (Sigma-Aldrich, 50 ng/ml), ionomycin (Enzo, 1 µg/ml), monensin (Enzo, 2 µg/ml). After 5 h, cells were stained with antibodies against surface markers, fixed, permeabilized, and stained with anti-IFN-γ mAb (XMG1.2, eBioscience), anti- IL-17 mAb (eBio17B7, eBioscience), or anti-TGF-β mAb (TW7-16B4, eBioscience) according to the Intracellular Staining Kit (Invitrogen) instructions. Flow cytometry was performed using the BD FACSCanto II (Becton Dickinson) and data were analyzed using FlowJo software (Treestar).
Tian J., Hong Y., Zhu Q., Zhou H., Zhang Y., Shen Z., Guo H., Zhang Y., Ai X., Zhao F., Rui K., Xu H, & Wang S. (2020). Mesenchymal Stem Cell Enhances the Function of MDSCs in Experimental Sjögren Syndrome. Frontiers in Immunology, 11, 604607.
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6

Isolation and Characterization of Intestinal CX3CR1+ Cells

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Following bacterial challenge, luminal contents were carefully recovered by gently flushing the intestine with PBS. Intraluminal CX3CR1+/gfp cells were isolated and characterized by flow cytometry as described in details elsewhere (5 (link)). Samples were analysed by BD FACSAria II (BD Biosciences). The following antibodies were used: CD11c (HL3) (BD Biosciences), CD103 (M290) (BD Biosciences), CD103 (2E7) (eBioscience), F4/80 (BM8) (eBioscience), MHC II (M5/114.15.2) (eBioscience), SiglecF (E50-2440) (BD Biosciences). For the isolation of CX3CR1+ cells intestinal tissue from CX3CR1+/gfp mice were collected and tissues repeatedly treated with HBSS containing EDTA (2mM). After each treatment tissues were shaken and supernatant discarded. After each wash an aliquot from the supernatant was analysed by microscopy to detect the presence of IECs; EDTA treatment was stopped (usually after 3-4 treatments) when epithelial cells were not present in the supernatant. Tissues were then treated for 50 minutes in RPMI 1640 with 10% FCS, 0.24mg/ml collagenase VIII (Sigma) and 40 U/ml DNase I (Roche) as described by others (19 (link)) ; after shaking cells suspensions were filtered and then purified by gradient separation as described before (5 (link)). Cells were sorted (>95% purity), suspended in PBS and injected into the intestinal lumen for pathogen exclusion assay.
Man A.L., Gicheva N., Regoli M., Rowley G., De Cunto G., Wellner N., Bassity E., Gulisano M., Bertelli E, & Nicoletti C. (2016). CX3CR1+ cell-mediated Salmonella-exclusion protects the intestinal mucosa during the initial stage of infection. Journal of immunology (Baltimore, Md. : 1950), 198(1), 335-343.
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7

Comprehensive Immune Cell Profiling

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In addition to critical reagents listed throughout this section, the following antibodies were used for flow cytometry and confocal imaging: anti–mouse CD45 (30-F11; BD Biosciences), CD3 (17A2; BioLegend), TCR Vγ5 (536; BioLegend), MHCII (M5/114.15.2; eBioscience), F4/80 (BM8; BioLegend), CD64 (X54-5/7.1; BioLegend), CD11b (M1/70; BioLegend), and Ly6C (AL-21; BD Biosciences).
Gentek R., Ghigo C., Hoeffel G., Jorquera A., Msallam R., Wienert S., Klauschen F., Ginhoux F, & Bajénoff M. (2018). Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult. The Journal of Experimental Medicine, 215(12), 2994-3005.
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8

Multiparametric Flow Cytometry Immunophenotyping

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The cells were incubated with FcR Blocking Reagent (Miltenyi Biotec, Auburn, CA) and Via-Probe (BD Biosciences) for 5 min followed by staining with specific antibodies for 30 min at 4 °C. Flow cytometry analyses were conducted on a FACSCanto II (BD Biosciences), and the data were analyzed with CellQuest Pro (BD Biosciences). The antibodies used were APC, FITC or PE-conjugated mAbs against CD11b (3A33) from Beckman Coulter (Brea, CA), F4/80 (BM8) and MHCII (M5/114.15.2) from eBioscience.
Hayashi S., Hamada T., Zinsou D.G., Oshiro M., Itoi K., Yamamoto T, & Kadowaki M. (2017). PI3K p85α Subunit-deficient Macrophages Protect Mice from Acute Colitis due to the Enhancement of IL-10 Production. Scientific Reports, 7, 6187.
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9

Multiparametric Flow Cytometry Analysis

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Single cell suspensions were incubated with a fixable viability dye (eBioscience) for 20 min at 4°C. Samples were blocked with FC block (24G2 grown in house and mouse serum) for 20 min followed by antibody staining for 20 min. Antibodies used: CD45.1 (A20, eBioscience), CD4 (RM4-5, eBioscience), Va2 (B20.1, BD), MHC II (M5/114.15.2, eBioscience), CD64 (X54-5/7.1, BioLegend), CD8a (53-6.7, eBioscience), CD103 (M290, BD Horizon), Ly6G (1A8 BD), CD69 (H1.2F3, BD), S1PR1 (713412, R&D Systems), interferon-γ (IFN-γ) (XMG1.2, BioLegend), and CD44 (IM7, eBioscience). Samples were washed twice with FACS buffer and acquired on a Miltenyi Macsquant analyzer. Samples were analyzed using FlowJo (Treestar) version 9.7.5.
Jaigirdar S.A., Benson R.A., Elmesmari A., Kurowska-Stolarska M.S., McInnes I.B., Garside P, & MacLeod M.K. (2017). Sphingosine-1-Phosphate Promotes the Persistence of Activated CD4 T Cells in Inflamed Sites. Frontiers in Immunology, 8, 1627.
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10

Flow Cytometry Immunophenotyping Procedure

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Example 7

To prevent non-specific binding of immunoglobulins to Fc receptor, the cells used in the following Examples were treated with CD16/32 (2.4G2) and stained with the following monoclonal antibodies: CD4 (RM4-5), CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), CD11b (M1/70), CD11c (N418), and MHCII (M5/114.15.2), from eBioscience; CD3e (145-2C1), and TCRγδ (GL3), from BD; CXCR3 (CXCR3-173), from Biolegend; and Live/Dead (Life technologies). All samples were analyzed using an LSR Fortessa (BD) and FlowJo software (Tree Star).

US11708399B2. Pharmaceutical composition comprising immunoglobulin Fc-fused interleukin-7 fusion protein for preventing or treating human papillomavirus-caused diseases (2023-07-25). GENEXINE, INC. [KR]. Inventors: Moon Cheol Kang [KR], Young Woo Choi [KR], Donghoon Choi [KR], Young Chul Sung [KR].
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