Cd45r b220

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CD45R/B220 is a cell surface antigen expressed on B cells and a subset of T cells. It is commonly used as a marker for identifying and enumerating B lymphocytes in flow cytometry applications.

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12 protocols using cd45r b220

Immunofluorescence Staining of OSCC

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Frozen sections were fixed in 2% paraformaldehyde/PBS pH 7.4 and blocked with 10% goat serum, 2% BSA, 0.02% fish skin gelatin and 0.05% TritonX100 (Sigma) in PBS for 1 h at room temperature. Paraffin sections of human OSCC were subjected to heat-mediated antigen retrieval (citrate buffer, pH6) prior to blocking. Primary antibodies were incubated overnight at 4 °C, followed by 1 h incubation at room temperature in secondary antibody.
The following primary antibodies were used: Foxp3 (eBioscience, clone FJK-16s, 1/100, and Abcam, clone 236 A/E7, 1/50), anti-Loricrin, anti-Krt76 (Santa Cruz, clone F-12, 1/100, and Sigma, HPA019656, 1/100), Krt14 (Covance, PRB-155P, 1/1000), B220/CD45R (eBioscience, clone RA3-6B2, 1/100), CD3 (BD Pharmingen, clone 17A2, 1/150), CD45 (BD Pharmingen clone 30-F11, 1/150); and secondary antibodies: anti-goat, anti-mouse and anti-rabbit Alexa Fluor 488, 568 and 633 (Life Technologies, 1/300).
EdU staining was performed with a Click-it EdU imaging kit (Life Technologies) according to the manufacturer’s recommendations. DAPI (Life Technologies) was used as a nuclear counterstain. Slides were mounted using ProLong Gold anti-fade reagent (Life Technologies). Images were acquired with a Nikon A1 Upright Confocal microscope. Images were analysed using ICY image analysis software^{55 (link)}.

Sequeira I., Neves J.F., Carrero D., Peng Q., Palasz N., Liakath-Ali K., Lord G.M., Morgan P.R., Lombardi G, & Watt F.M. (2018). Immunomodulatory role of Keratin 76 in oral and gastric cancer. Nature Communications, 9, 3437.

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Multiparameter Flow Cytometric Analysis of Mouse B Cells

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Monoclonal antibodies to the following mouse E-proteins were used in multiparameter flow cytometric analysis: B220/CD45R (RA3-6B2), CD19 (eBio1D3), CD86 (GL1), CXCR4/CD184 (2B11), and GL7 (GL7) from eBioscience; CD138/Syndecan-1 (281-2), Fas/CD95 (Jo2), and IgG1 (X56) from BD; and peanut agglutinin (PNA) from Vector Laboratories. Antibodies to the following proteins were generated and conjugated in-house: Bcl6 (7D1-10), CD21 (7G6), CD23 (B3B4), CD38 (NIMR5), CD138/Syndecan-1 (7/11-8B8), Gr1/Ly6c (RB6-8C5), IgD (11-26C), and IgM (331.12). All antibodies were titrated for optimum concentration before use, with dilutions ranging from 1/100 to 1/1,600. FcγRII/III (24G2; supernatant) was used for blocking. Biotinylated monoclonal antibodies were detected with streptavidin conjugated to PECy7 (BD). NP was conjugated in-house. Viable cells were identified by propidium iodide or SytoxBlue (Invitrogen) exclusion. For intracellular staining, cells were fixed, permeabilized, and stained using the reagents and protocol in the Foxp3 Staining Buffer Set (eBioscience). Cells were analyzed on FACSCanto II or LSR Fortessa cytometers (BD). Cells were sorted on a FACS Aria cytometer (BD). Data were processed using FlowJo software.

Gloury R., Zotos D., Zuidscherwoude M., Masson F., Liao Y., Hasbold J., Corcoran L.M., Hodgkin P.D., Belz G.T., Shi W., Nutt S.L., Tarlinton D.M, & Kallies A. (2016). Dynamic changes in Id3 and E-protein activity orchestrate germinal center and plasma cell development. The Journal of Experimental Medicine, 213(6), 1095-1111.

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Comprehensive Murine B Cell Immunophenotyping

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Single-cell suspensions of BM, Spleen, and of blood cells were obtained from 8-16 week old mice. Depletion of erythrocytes from the various sample was achieved using Red Blood Cell Lysis Buffer (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Next, the cells were immunostained with a relevant mixture of flourochrome conjugated mAbs against any of the following selected mouse B cell surface antigens B220/CD45R, CD19, CD43, HSA/CD24, CD25, AA4.1/CD93, CD23, IgM, and IgD; all obtained from eBioscience Inc. (San Diego, CA). Next, by polychromatic flow cytometry we analyzed the samples for relevant B cell developmental stages in the BM and periphery according to the Hardy classification and the Philadelphia nomenclature [26 (link), 42 (link)]. The following B cells stages were identified: pro-B/Fr. B-C (B220⁺ CD25⁻ CD43⁺ CD24⁺⁺ CD19^+/++ CD93⁺); pre-B/Fr. D (B220⁺⁺ CD25⁺ CD43⁻ CD24⁺⁺ CD19⁺⁺⁺ CD93⁺ IgM⁻); immature/Fr. E (B220⁺⁺⁺ CD43+ CD24^hi CD19⁺⁺⁺ CD93⁺ CD25⁻/IgM⁺ IgD⁻); mature recirculating/Fr. F (B220⁺⁺⁺ CD19⁺⁺⁺ CD23^++/hi CD93⁻); and splenic transitional T2 plus follicular (B220⁺⁺⁺ CD19⁺⁺⁺ CD23^++/hi). Data were acquired on a FACSAria instrument (BD Biosciences, San Jose, CA), and analyzed/plotted using the FlowJo V.10 software (FlowJo, Ashland, OR).

Marcu-Malina V., Goldberg S., Vax E., Amariglio N., Goldstein I, & Rechavi G. (2016). ADAR1 is vital for B cell lineage development in the mouse bone marrow. Oncotarget, 7(34), 54370-54379.

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Multiparameter Flow Cytometry Analysis

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Cell suspensions were obtained from intestines, spleens, and MLNs and stained in PBS 2% FCS with conjugated antibodies to the following markers: TCR-β (eBioscience), TCR-γδ (eBioscience), CD3 (eBioscience), CD45 (eBioscience), CD4 (eBioscience), CD8a (eBioscience), CD62L (eBioscience), CD44 (BD), CD45R (B220; eBioscience), CD357 (GITR; eBioscience), CCR9 (eBioscience), α4β7 (BioLegend), Foxp3 (eBioscience), IgA (eBioscience), IgM (eBioscience), CD25 (eBioscience), CD103 (eBioscience), CD45RB (BioLegend), and Nrp-1 (R&D Systems). Live/dead cell discrimination was obtained using the Aqua Dead Cell Stain kit (Invitrogen). For cytokine production, cells were stimulated for 4 h with 20 ng/ml PMA (Sigma-Aldrich) and 0.5 µg/ml ionomycin (Sigma-Aldrich). Golgi Stop (1,000×; BD) was added during the last 3 h of stimulation. Cells were fixed and permeabilized using intracellular fixation and permeabilization buffer kit (eBioscience) and stained for conjugated anti–IL-17A (eBioscience) and anti–IFN-γ (eBioscience). Flow cytometry data were acquired at FACSCanto II. Data were analyzed with FlowJo software (version 7.6.5).

Rigoni R., Fontana E., Guglielmetti S., Fosso B., D’Erchia A.M., Maina V., Taverniti V., Castiello M.C., Mantero S., Pacchiana G., Musio S., Pedotti R., Selmi C., Mora J.R., Pesole G., Vezzoni P., Poliani P.L., Grassi F., Villa A, & Cassani B. (2016). Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects. The Journal of Experimental Medicine, 213(3), 355-375.

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Assessing Immune Cell Apoptosis and Oxidative Stress

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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies, and an Annexin V-FITC apoptosis kit were purchased from eBioscience (San Diego, CA, USA). The 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), α-lipoic acid, and 5-methoxytryptamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3534 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-γH2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA).
The 5-methoxytryptamine-α-lipoic acid (MLA) was synthetized using α-lipoic acid and 5-methoxytryptamine at the drug department of the Institute of Radiation Medicine, CAMS. Melatonin (MLT) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). MLA and MLT were dissolved in a 5% carboxymethyl cellulose (CMC) solution.

Li D., Tian Z., Tang W., Zhang J., Lu L., Sun Z., Zhou Z, & Fan F. (2016). The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury. International Journal of Molecular Sciences, 17(6), 935.

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Multicolor Flow Cytometric Analysis of Hematopoietic Cells

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Anti-mouse-CD45.1-FITC, CD45.2-PE, Gr-1-PE/CY7, CD45R/B220-PE/CY5.5 CD11b-PE/CY7 and CD3-APCwere purchased from BioLegend (San Diego, CA, USA); anti-mouse-Sca-1-PE, CD117 (c-kit) Alexa Fluor 700, CD4, CD8, CD45R/B220, Gr-1, CD11b, Ter119 and APC/CY7-conjugated streptavidin were purchased from eBioscience (San Diego, CA, USA); iron-dextrin was purchased from Pharmacosmos A/S (Denmark); the ROS staining kit (S0033) and NAC were purchased from the Beyotime Institute of Biotechnology; calcein-AM fluorescent dye was purchased from Sigma-Aldrich (USA); deferasirox was purchased from Novartis; RPMI 1640 was purchased from Gibco (USA); methylcellulose M3434 was purchased from Stem cell (USA); fetal calf serum was purchased from Bioind (Italy); CD117 MicroBeads (130-091-229) were purchased from Miltenyi Biotec (Germany); a RNA PCR Kit (AMV) Ver.3.0 (DRR019A) was purchased from Takara; a RNeasy MicroKit (74004) was purchased from Qiagen (Germany); the NOX4 and GPX1 gene qRT-PCR primers were synthesized by Sangon Biotech (Shanghai, China).

Chai X., Li D., Cao X., Zhang Y., Mu J., Lu W., Xiao X., Li C., Meng J., Chen J., Li Q., Wang J., Meng A, & Zhao M. (2015). ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice. Scientific Reports, 5, 10181.

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Multicolor Cytometry Analysis of Immune Cells

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Cytofluorimetry analysis was performed as previously described (16 (link)). In brief, single-cell suspensions from muscle or spleen were stained with MAbs against the following antigens: CD45 as a pan leukocyte (clone OX-1), TCRαβ (clone R7/3), CD45RA on B cells (clone OX33), CD45R/B220 on B cells (clone His24), anti-granulocytes (RP-1 and His48), CD4 (clone w3/25), CD45RC (clone OX22 or clone OX32), CD25 (clone OX39), CD8 (clone OX8), CD172a/SIRPα (clone OX41), CD161 on NK and myeloid cells (clone 3.2.3), CD163 on macrophages (clone ED2), CD68 for macrophages (clone ED1), and with viability dye eFluor506 or eFluor450 from eBiosciences to assess cell viability. Analysis was performed on a BD FACS Verse with FACSuite Software version 1.0.6. Post-acquisition analysis was performed using FlowJo software. The CD45RC⁻ population was defined using an isotype control. The CD45RC^high population was defined using as reference the levels of CD45RC expression on B cells since they always express the highest levels of CD45RC. The CD45RC^low population corresponded to the intermediate population between the CD45RC⁻ and the CD45RC^high cells.

Ouisse L.H., Remy S., Lafoux A., Larcher T., Tesson L., Chenouard V., Guillonneau C., Brusselle L., Vimond N., Rouger K., Péréon Y., Chenouard A., Gras-Le Guen C., Braudeau C., Josien R., Huchet C, & Anegon I. (2019). Immunophenotype of a Rat Model of Duchenne's Disease and Demonstration of Improved Muscle Strength After Anti-CD45RC Antibody Treatment. Frontiers in Immunology, 10, 2131.

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Flow Cytometric Analysis of Lung and Spleen Cells

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Lung and spleen tissue cells were analyzed using flow cytometry as described previously^{24 (link)}. Briefly, cell samples were incubated in staining buffer with 10% goat serum and 5 µg/mL anti-CD16/CD32 to block nonspecific binding. Antibodies to IL-13, IFN-γ, FoxP3, CD11c, MHC class II, CD4, CD11b, and CD45R/B220 were purchased from eBioscience (San Diego, CA). Antibodies to CD103, CD40, Siglec-F, CD86, and CD45 were from BD Biosciences (San Jose, CA). Anti-neutrophil antibody (7/4) was purchased from Abcam (Cambridge, MA). SHIP-1 antibody (P1C1) was from Santa Cruz Biotechnology (Dallas, TX) and conjugated to PE or Alexa Fluor 647 (AbLab, Vancouver, British Columbia, Canada). ILC2 cells in lung and spleen were identified as Lineage⁻B220⁻CD127⁺CD25⁺CD90.2⁺T1/ST2⁺ cells, which are primarily Sca-1⁺ and CD117⁺. Intracellular staining was performed as described previously with modifications^{24 (link)}. Dead cells were excluded using eFluor fixable viability dyes (eBioscience). Samples were acquired on a BD LSR II Flow Cytometer, and data analysis was performed using the FlowJo software (Tree Star, Ashland, OR).

Ye X., Zhang F., Zhou L., Wei Y., Zhang L., Wang L., Tang H., Chen Z., Kerr W.G., Zheng T, & Zhu Z. (2021). Selective deletion of SHIP-1 in hematopoietic cells in mice leads to severe lung inflammation involving ILC2 cells. Scientific Reports, 11, 9220.

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Multiparameter Immune Cell Analysis

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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies were purchased from eBioscience (San Diego, CA, USA). The 21, 71-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich (St Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3434 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-H2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA). Hei brick tea was purchased from Baishaxi Tea Industry (Hunan, China). Malondialdehyde (MDA), SOD, CATand GSHPx reagent kits were purchased from Nanjing Jiancheng Bioengineering (Jiangsu, China).

Long W., Zhang G., Dong Y, & Li D. (2018). Dark tea extract mitigates hematopoietic radiation injury with antioxidative activity. Journal of Radiation Research, 59(4), 387-394.

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Multicolor Flow Cytometry Analysis of Murine Splenocytes

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Spleen single-cell suspensions were incubated with a Fc-receptor blocking antibody 2.4G2 (kindly provided by Dr. Louis Boon, Bioceros BV, Utrecht, The Netherlands) and the percentage of spleen cell populations (macrophages, B cells, Dendritic cells, T cells, monocytes) were determined using the following antibodies: F4/80 (BM8) (Invitrogen, Bleiswijk, The Netherlands), CD45R (B220) (eBioscience, Vienna, Austria), CD11c (HL3) (BD Biosciences), CD4 (GK1.5) (eBioscience, Vienna, Austria), CD8α (53-6.7) (eBioscience, Vienna, Austria), CD3e (145-2C11) (eBioscience, Vienna, Austria), Lineage (Lin) (B220, NK1.1, CD90, CD49. Ly6G) (ebioscience, Vienna, Austria) CD45.1 (ebioscience, Vienna, Austria). Samples were analyzed with a LSR Fortessa II (Beckman Coulter) and the FlowJo software (Tree Star Inc., Ashland, The United States). The different spleen cell populations were defined as follow: DCs (CD11c+MHCII+), macrophages (F4/80+), B cells (CD45R+MHCII+), CD4+ (CD3+CD8-CD4+), CD8+T cells (CD3+CD8+CD4-) and monocytes (CD45+Lin-F4/80-CD11c-MHCII-CD11b+Ly-6C+). Percentages were reported to the total number of splenocytes of each mouse to calculate the number of cells per population (Figure S2 and S3).

Costes L.M., van der Vliet J., Farro G., Matteoli G., van Bree S.H., Olivier B.J., Nolte M.A., Boeckxstaens G.E, & Cailotto C. (2014). The Spleen Responds to Intestinal Manipulation but Does Not Participate in the Inflammatory Response in a Mouse Model of Postoperative Ileus. PLoS ONE, 9(7), e102211.

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