Cd45.2 104

Manufactured by Thermo Fisher Scientific

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The CD45.2 (104) is a laboratory instrument used for the detection and analysis of the CD45.2 antigen. It is a commonly used marker in immunology research and flow cytometry applications.

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CD45.2 (clone 104, (14 citations) Anti-CD45.2 (104) (9 citations) Anti-CD45.2 (clone 104, (7 citations) Anti-CD45.2-PE (clone 104) monoclonal antibodies (3 citations) Anti-CD45.2-PE (clone 104) (3 citations) CD45.2-APC (104) (2 citations) APC-Cy7 conjugated anti-CD45.2 (clone 104, (2 citations) Anti-CD45.2-biotin (104; (2 citations) CD45.2-FITC (clone 104) (2 citations)

13 protocols using cd45.2 104

Multiparametric Analysis of Intestinal Immune Cells

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Intestinal tissues were fixed in 4% paraformaldehyde, rehydrated in 20% sucrose, and frozen in OCT media (Sakura). Tissues were cut into 7–8μm sections and treated with ice cold acetone. Sections were treated with biotin-avidin blocking (Vector labs) and stained with the following biotinylated or directly conjugated antibodies:
CD8β (YTS156.7.7, Biolegend), CD45.2 (104, eBioscience), Epcam (G8.8, Biolegend), CD11b (M1/70, eBioscience), and CD11c (HL3, eBioscience). For identification of YFP-producing cells, sections were stained with anti-GFP (Abcam, ab6556), anti-rabbit-FITC (Abcam, ab6108), and anti-FITC-AlexaFlour488 (Invitrogen, polyclonal) antibodies. No reactivity was observed in infected YFP-negative mice. Stained slides were mounted with Prolong Gold antifade reagent (Thermo Fisher Scientific), imaged using a Nikon 90i, and analyzed using Adobe Photoshop software.

Bergsbaken T., Bevan M.J, & Fink P.J. (2017). Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells. Cell reports, 19(1), 114-124.

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Hematopoietic Stem Cell Transplantation Assay

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GFP⁺ or GFP^– LSK cells from Evi1-GFP mice were sorted and 100, 500, or 2500 GFP⁺ or GFP^– cells were subsequently transplanted into groups of SJL (CD45.1⁺) congenic mice hosts (three mice/dose). Host mice were lethally irradiated using a Cs-137 Irradiator in two equal doses of 500 rads separated by at least 2 h. Cells were injected into the retro-orbital venous sinus of anesthetized recipient mice. All transplanted hosts are subsequently maintained on antibiotic water. Beginning 4 weeks after transplantation and continuing for at least 16 weeks, peripheral blood was collected from the tail veins of recipient mice and analyzed by FCM for the lineage markers B220 (6B2), Mac-1 (M1/70), CD4 (L3T4) and CD8 (Ly-3), and Gr-1 (8C5) to monitor engraftment. Donor and host cells were distinguished by expression of CD45.1 (A20, eBioscience) and CD45.2 (104, eBioscience) to determine the level of chimerism.

Wang Y., Tian H., Cai W., Lian Z., Bhavanasi D., Wu C., Sato T., Kurokawa M., Wu D., Fu L., Wang H., Shen H., Liang D, & Huang J. (2018). Tracking hematopoietic precursor division ex vivo in real time. Stem Cell Research & Therapy, 9, 16.

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Multiparametric T Cell Analysis

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Thymus and spleen were mechanically teased with glass slides to acquire single cell suspensions and cells were stained with antibodies to the following: CD45.1 (A20, eBioscience), CD45.2 (104, eBioscience), CD4 (RM4‐5, Biolegend), CD8 (53‐6.7, Biolegend), CD25 (PC61.5, Biolegend), CD44 (IM7, eBioscience), Qa2 (695H1‐9‐9, Biolegend), TCRβ (H57‐597, eBioscience), CD69 (H1.2F3, eBioscience), and H‐2K^b (AF6‐88.5, Biolegend). Reagents were conjugated to Pacific Blue, Brilliant Violet (BV) 421, BV510, BV711, PE, PE‐Cy7, PerCP–eFluor 710, allophycocyanin– eFluor 780 and Alexa Fluor 700. Streptavidin PE‐Cy7 (eBioscience) was used to detect biotinylated antibodies. A Foxp3 fixation kit (eBioscience) was used in conjunction with anti‐Foxp3 (FJK‐16s, eBioscience) or the BD Cytofix/Cytoperm Kit (BD Biosciences) to preserve the GFP signal. Data were acquired using a BD LSR Fortessa and FACSDiva 2.6 software and analysed using FloJo software (Tree Star).

Cowan J.E., Baik S., McCarthy N.I., Parnell S.M., White A.J., Jenkinson W.E, & Anderson G. (2018). Aire controls the recirculation of murine Foxp3+ regulatory T‐cells back to the thymus. European Journal of Immunology, 48(5), 844-854.

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Laminin Isoforms and Cell Markers Analysis

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The antibodies used in immunofluorescence staining and flow cytometry were laminin α4 (377; Ringelmann et al., 1999 (link)), laminin α5 (4G6; Sorokin et al., 1997 (link)), laminin γ1 (3E10; Sixt et al., 2001a (link)), pan-laminin (455; Sorokin et al., 1997 (link)), CD45 (30G.12), CD45.2 (104, eBioscience), CD45.1 (A20, BD PharMingen), CD11b/MAC-1 (M1/70, BD PharMingen), CD11c (N418, eBioscience), CD4 (H129.19, BD PharMingen), CD8 (53-6.7, eBioscience), B220 (RA3-6B2, BD PharMingen), IL-17 (TC11-18H10.1, BD PharMingen), IFN-γ (XMG1.2, BD PharMingen), integrin β1 (eBioHMb1-1 [HMb1-1], eBioscience), integrin β2 (C71/16, BD PharMingen), integrin β3 (2C9.G2, BD PharMingen), integrin α6 (GoH3, PharMingen), integrin α5 (5H10-27 [MFR5], BD PharMingen), integrin αv (RMV-7, BD PharMingen), MCAM (ME-9F1, Miltenyi Biotec), and integrin α4 (PS/2, BD PharMingen).
Laminin 411 and 511 were purified as previously described (Sixt et al., 2001b (link)). Recombinant laminin α5 domain IVa was produced in HEK293 cells as described previously (Sasaki and Timpl, 2001 (link)); mini-laminin 511 composed of the C-terminal sequences of laminin α5, β1, and γ1 chains was from Takara Bio (Künneken et al., 2004 (link); Miyazaki et al., 2008 (link)).

Zhang X., Wang Y., Song J., Gerwien H., Chuquisana O., Chashchina A., Denz C, & Sorokin L. (2020). The endothelial basement membrane acts as a checkpoint for entry of pathogenic T cells into the brain. The Journal of Experimental Medicine, 217(7), e20191339.

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Isolating Lung Immune Cells

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The left lungs were dissected and digested with Collagenase type 4 (Sigma-Aldrich; 0.5 mg/ml) at 37°C for 40 min. A syringe plunger (BD Bioscience) was used to disperse the cells. Cell suspensions were then filtered with a cell strainer (Corning Inc., USA) and incubated with ACK lysing Buffer (Thermo Fisher Scientific) at room temperature for 1 min to deplete erythrocytes. Cells (1 × 10⁶) were stained using appropriate combinations of FITC-, PercP-, Pecy7-, and APC-labeled monoclonal antibodies to CD11b (M1/70), CD45.1 (A20), Ly6G (1A8) (BD Pharmingen, USA), and CD45.2 (104) (eBioscience). Cells were fixed with 2% paraformaldehyde and analyzed by flow cytometry (FACSCanto, BD Biosciences) using the FlowJo software.

Bomfim C.C., Amaral E.P., Cassado A.D., Salles É.M., do Nascimento R.S., Lasunskaia E., Hirata M.H., Álvarez J.M, & D’Império-Lima M.R. (2017). P2X7 Receptor in Bone Marrow-Derived Cells Aggravates Tuberculosis Caused by Hypervirulent Mycobacterium bovis. Frontiers in Immunology, 8, 435.

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Immunophenotyping of Mouse Leukocytes

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We stained mouse leukocytes with monoclonal antibodies to CD3 (17A2; BioLegend), TCRβ (H57-597; eBioscience), TCRγ/δ (GL3; BioLegend), CD5 (53-7.3; eBioscience), CD19 (6D5; BioLegend), CD11b (M1/70; BD Pharmingen), CD11c (N418; eBioscience), NK1.1 (PK136; eBiosciences), CD45.2 (104; eBioscience), Thy1.2 (53-2.1; BioLegend), CD8a (53-6.7, BD Pharmingen), CD4 (RM4-5; BD Pharmingen), RORγt (B2D; eBioscience), Gata-3 (TWAJ; eBioscience), c-kit (2B8; eBioscience), IL-7Rα (A7R34; eBioscience), Siglec-F (E50-2440; BD Pharmingen) and Fixable Viability Dye eFluor780 (eBioscience). All samples were acquired with an LSR II (Becton Dickinson) flow cytometer and analyzed with FlowJo software (Treestar Inc.).

Frascoli M., Jeanty C., Fleck S., Moradi P.W., Keating S., Mattis A.N., Tang Q, & MacKenzie T.C. (2016). Heightened immune activation in fetuses with gastroschisis may be blocked by targeting IL-5. Journal of immunology (Baltimore, Md. : 1950), 196(12), 4957-4966.

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Multiparameter Flow Cytometry Panel

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Fluorescein-conjugated mAbs specific for the mouse antigens, CD3 (145-2C11), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), IA/IE (MKS4), CD80 (16-10A1), CD86 (GL1), CD40 (1C10), CD25 (PC61.5), CD69 (H1.2F3), Vβ5.1/5.2 (MR9-4), CD45.1 (A20), and CD45.2 (104) were purchased from eBioscience (San Diego, CA). An IL-10 neutralizing mAb was purchased from R&D Systems (Minneapolis, MN). For cell surface staining, cells were incubated with fluorescence-conjugated mAbs in the presence of 2.4G2 or rat sera. Matched isotype controls were used to establish background fluorescence. 7-AAD was used to exclude dead cells in phenotype analysis. For cell counting, stained cells were collected at high speed for 40 sec and counted via flow cytometer. Phenotype analysis and cell counting were performed on a BD FACSAria (BD Biosciences, Sand Jose, CA) using the BD FACSDiVa software (BD Biosciences).

Wang Q., He H., Chen D., Wang C., Xu Y, & Song W. (2017). Hepatic stroma-educated regulatory DCs suppress CD8+ T cell proliferation in mice. Oncotarget, 8(55), 93414-93425.

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Multiparameter Flow Cytometry of Immune Cells

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Single cells were re-suspended in phosphate buffered saline (PBS), 2% fetal bovine serum (FBS), and stained with fluorochrome-conjugated or biotinylated antibodies against B220 (RA3-6B2), CD19 (1D3), CD24 (M1/69), CD3 (145-2C11), CD4 (GK1.5 or RM4-5), CD5 (53–7.3), CD8 (53–6.7), CD11c (HL3), CD11b (M1/70), CD117 (2B8), Ter119, CD184 (CXCR4, 2B11), CXCR3 (CXCR3-173), CCR7 (4B12), CXCR5 (2G8), CD11a (M17/4), CD49d (9C10, MFR4.B), CD54 (3E2), CD62L (MEL-16), NK1.1 (PK136), TCRγδ (GL3), TCRβ (H57-597), Ly6G (1A8), Ly6C (AL-21), CD278 (7E.17G9), CD279 (RMP1-30), CD44 (IM7), CD25 (PC61), CD45RB (16A), Qa2 (695H1-9-9), CD69 (H1.2.F3), Bcl-6 (BCL-DWN), CD278 (ICOS), CD45.1 (A20), or CD45.2 (104) (all from eBioscience, Biolegend, or BD Pharmingen). Biotin-labeled antibodies were visualized with fluorochrome-conjugated streptavidin (eBioscience). The intracellular staining of Bcl-6 was performed as described in the manufacturer’s protocol. LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (ThermoFisher) was used in all experiments to exclude dead cells. Compensation was performed using CompBeads (BD Biosciences) and ArC^TM Amine Reactive Compensation Bead individually stained with each fluorochrome. Compensation matrices were calculated with FACSdiva software. Data acquisition was done on FACSCanto II (BD) flow cytometer and analyzed with FlowJo software version 9 (Treestar).

Hwang I.Y., Harrison K., Park C, & Kehrl J.H. (2017). Loss of Gαi proteins impairs thymocyte development, disrupts T-cell trafficking, and leads to an expanded population of splenic CD4+PD-1+CXCR5+/− T-cells. Scientific Reports, 7, 4156.

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Monoclonal Antibodies for Cell Identification

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The monoclonal antibodies used in this study targeted the following proteins: CRLR (H-42; Santa Cruz, Dallas, TX, USA), RAMP-1 (EPR10867; abcam, Cambridge, UK), Gr-1 (RB6-8C5; BioLegend, San Diego, CA, USA), Mac-1 (M1/70; BioLegend), B220 (RA3-6B2; BioLegend), CD3e (145-2C11; Biolegend), CD2 (RM2-5; TONBO, Japan), CD8a (53–6.72; TONBO), TER-119 (TER-119; TONBO), CD127 (A7R34; TONBO), c-Kit (2B8; BioLegend), Sca-1 (D7; BioLegend), CD34 (RAM34; eBioscience, Santa Clara, CA, USA), CD16/32 (93; eBioscience), CD48 (HM48-1; BioLegend), CD150 (TC15–12F12.2; BioLegend), CD45.2 (104; eBioscience) and CD45.1 (A20; eBioscience). A mixture of mAbs against CD2, CD3e, CD8a, B220, TER-119, CD127, Mac-1 and Gr-1 served as lineage markers (Lineage). Mouse anti-BrdU-Alexa Fluor 647 antibody (3D4; BD Biosciences) was used to detect intracellular BrdU.

Suekane A., Saito Y., Nakahata S., Ichikawa T., Ogoh H., Tsujikawa K, & Morishita K. (2019). CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis. Scientific Reports, 9, 429.

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Flow Cytometry Immunophenotyping Protocol

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The following antibodies were used for flow cytometry: CD3e (145-2C11; eBioscience and 500A2; BD), CD4 (RM4-5; BD), CD45.2 (104; eBioscience), CD90.1 (HIS51; eBioscience). Intracellular cytokine staining was performed with anti-IFNγ (XMG1.2; BD) or anti-TNFα (MP6-XT22; BD), using the Cytofix/Cytoperm kit (BD Biosciences). Flow cytometric data were collected on an LSR II (BD Biosciences) and analyzed with FlowJo software (TreeStar).

Torabi-Parizi P., Vrisekoop N., Kastenmuller W., Gerner M.Y., Egen J.G, & Germain R.N. (2014). Pathogen-related differences in the abundance of presented antigen are reflected in CD4+ T cell dynamic behavior and effector function in the lung. Journal of immunology (Baltimore, Md. : 1950), 192(4), 1651-1660.

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