Cd45.2 fitc

Manufactured by Thermo Fisher Scientific

CD45.2-FITC is a fluorescently-labeled antibody that targets the CD45.2 antigen, which is expressed on the surface of certain immune cells. This product can be used in various flow cytometry applications to identify and analyze cell populations.

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Close spelling variants of this product

CD45-FITC (92 citations) CD45.2-FITC (clone 104) (2 citations) Anti-CD45.2-FITC ( (2 citations) CD45.1-FITC and CD45.2-PE antibodies (2 citations)

15 protocols using cd45.2 fitc

Characterizing T Cell Subsets in Mice

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Single cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR-

β

APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR-

β

PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD nearIR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-II or a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). Conventional CD4⁺ cells were identified as live TCR-

β

+ CD4⁺ CD25- NK1.1-, and then CD44 and CD62L were used to identify EM (CD44+CD62L-) and CM (CD44+CD62L+) subsets.

Hogan T., Nowicka M., Cownden D., Pearson C.F., Yates A.J, & Seddon B. (2019). Differential impact of self and environmental antigens on the ontogeny and maintenance of CD4+ T cell memory. eLife, 8, e48901.

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Isolation and Flow Cytometric Analysis of Murine Brain Immune Cells

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Mice were euthanized with CO₂ and perfused through the left ventricle with 20mL of PBS. Whole brain was removed and placed in 2.5 mL of digestion buffer (PBS, 5% FCS, 1mM HEPES) before finely chopping. 400U of Collagenase D (Roche) was added to the mixture, then incubated at 37°C for 30 min before adding 50 μL of 0.5M EDTA followed by a 5 min incubation. Digested tissue was mashed through a 40μm cell strainer, pelleted at 700g in a swinging bucket centrifuge, then resuspended in 10mL of 38% isotonic Percoll, and centrifuged at 2000 RPM for 30 min with no brake. The myelin debris layer was removed by aspiration and the pellet washed with PBS. Cells were then blocked with 1:100 FcX (BioLegend 156604) before a 15 min incubation with the following antibodies at a 1:200 dilution in PBS: CD45.2-FITC (eBioscience 104 cat #11-0454-82), CD4-BUV395 (BD GK1.5 cat #563790), CD11b-BV421 (BioLegend M1/70 cat #101235, CX3CR1-APC (BioLegend SA011F11 cat #149008). Cells were washed in PBS, then fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) before staining with PU.1-PE (BioLegend 7C2C34 cat #681307) or rat IgG2a-PE isotype (BioLegend RTK2758, cat #400507) for 30 minutes at room temperature. Cells were washed once, resuspended, run on a LSRFortessa cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

Chung H., Parkhurst C.N., Magee E.M., Phillips D., Habibi E., Chen F., Yeung B., Waldman J., Artis D, & Regev A. (2021). Joint single-cell measurements of nuclear proteins and RNA in vivo. Nature methods, 18(10), 1204-1212.

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Multiparameter Flow Cytometry Panel

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The following monoclonal antibodies and cell dyes were used: CD45.1 FITC, CD45.2 AlexaFluor700, CD45.2 FITC, TCR-beta APC, CD4 PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE-Cy7, L-selectin eFluor450, CD122 biotin (all eBioscience); CD8 Pacific Orange, streptavidin PE-TexasRed, LIVE/DEAD blue (all Invitrogen); and CD45.1 Brilliant Violet 650, CD4 Brilliant Violet 711, and TCR-beta PerCP-Cy5.5 (all BioLegend). Samples were acquired on LSR-II, LSRFortessa, or Fortessa X20 flow cytometers (BD), and analysis was performed with FlowJo software (Treestar).

Rane S., Hogan T., Seddon B, & Yates A.J. (2018). Age is not just a number: Naive T cells increase their ability to persist in the circulation over time. PLoS Biology, 16(4), e2003949.

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Multicolor Flow Cytometry Panel

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The following antibodies were used: mouse lineage cocktail-PE (BioLegend, cat# 78035), mouse lineage cocktail-APC (R&D Systems, cat# FLC001A), c-Kit-FITC (BioLegend, cat# 161603), c-Kit-APC (BioLegend, cat# 135108), c-Kit-PE/Cy7 (BioLegend, cat# 105814), c-Kit-BV421 (BioLegend, cat# 135124), Sca-1-BV605 (BioLegend, cat# 108133), Sca-1-APC (eBioscience, cat# 17-5981-82), Sca-1-BV421 (BioLegend, cat# 108127), Sca-1-PerCP-Cy5.5 (eBioscience, cat# 45-5981-82), CD150-BV421 (BioLegend, cat# 115925), CD150-PE (eBioscience, cat# 12-1501-82), CD150-PE-Cy7 (BioLegend, cat# 115913), CD48-PE/Cy7 (eBioscience, cat# 25-0481-80), CD48-BV711 (BioLegend, cat# 103439), CD48-APC-Cy7 (BioLegend, cat# 103432),CD34-FITC (eBioscience, cat# 11-0341-82), CD135-PE-Cy5 (BioLegend, cat# 135311), CD135-APC (BioLegend, cat# 135310), CD45.1-PerCP-Cy5.5 (BioLegend, cat# 110727), CD45.2-BV421 (BioLegend, cat# 109831), CD45.2-FITC(eBioscience, cat# 11-0454-82), Gr1-PE (BioLegend, cat# 108407), CD11b-APC (BioLegend cat# 101211), CD4-PE (BioLegend,cat# 116005), CD8a-PE (BioLegend, cat# 100707), B220-APC (BioLegend, cat# 103211).

Wang S., Han J., Huang J., Islam K., Shi Y., Zhou Y., Kim D., Zhou J., Lian Z., Liu Y, & Huang J. (2024). Deep learning-based predictive classification of functional subpopulations of hematopoietic stem cells and multipotent progenitors. Stem Cell Research & Therapy, 15, 74.

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Phenotypic analysis of lymphocyte subsets

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Single-cell suspensions were prepared from the thymus, spleen and lymph nodes of busulfan chimeric mice, wildtype control mice, or germ-free mice. Cells were stained with the following monoclonal antibodies and cell dyes: CD45.1 FITC, CD45.2 FITC, CD45.2 AlexaFluor700, TCR-

β

APC, CD4⁺ PerCP-eFluor710, CD44 APC-eFluor780, CD25 PE, CD25 eFluor450, CD25 PE-Cy7, CD62L eFluor450, NK1.1 PE-Cy7 (all eBioscience), CD45.1 BV650, CD45.2 PE-Dazzle, TCR-

β

PerCP-Cy5.5 CD4⁺ BV711, CD44 BV785, CD25 BV650 (all Biolegend), CD62L BUV737 (BD Biosciences), LIVE/DEAD near-IR and LIVE/DEAD blue viability dyes. For Ki67 staining, cells were fixed using the eBioscience Foxp3 /Transcription Factor Staining Buffer Set and stained with either anti-mouse Ki67 FITC or PE (both eBioscience). Cells were acquired on a BD LSR-Fortessa flow cytometer and analysed with Flowjo software (Treestar). See Figure 1—figure supplement 1 for the gating strategy used to identify mature single positive thymocytes and peripheral naive subsets, and gates to measure Ki67 frequencies.

Rane S., Hogan T., Lee E., Seddon B, & Yates A.J. (2022). Towards a unified model of naive T cell dynamics across the lifespan. eLife, 11, e78168.

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Immune Response Modulation Protocol

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7,12-dimethylbenz(a)anthracene (DMBA) (≥ 95% purity), N6, 2’-O-dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP) and Sodium orthovandate (Na3VO4) were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained from LC laboratories (Woburn, MA). Rat anti-mouse IL-12Rβ2 and IL-23R were purchased from R&D; Rabbit anti-mouse VEGF, TRP2, Mouse anti-human S100, Rat anti-mouse vimentin were obtained from Santa Cruz Biotechnology, Inc. Rat anti-mouse pERK was from BD biosciences. CD4-PE, CD4-FITC, FOXP3-PE, FOXP3-v450, IA/IE-FITC, IL-17-Percp-Cy5.5, IL-10-PE, CD45.2-Percp-Cy5.5, CD45.2-FITC were obtained from eBiosciences. IFNγ-PE-Cy7, CD8-Alexa-647, and CD8-PE were obtained from BD-pharmingen.

Nasti T.H., Cochran J.B., Vachhani R.V., McKay K., Tsuruta Y., Athar M., Timares L, & Elmets C.A. (2016). IL-23 Inhibits Melanoma Development by Augmenting DNA Repair and Modulating T-cell Subpopulations. Journal of immunology (Baltimore, Md. : 1950), 198(2), 950-961.

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Multicolor Flow Cytometry Antibody Panel

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Commercial antibodies and staining reagents, from BioLegend: GL7-PacBlue, GL7-PE, GL7-A647, B220-PerCP-Cy5.5, B220-APC-Cy7, IgMb-FITC, IgMa-PE, CD45.2-FITC, CD45.2-APC, CD45.1-FITC, CD45.1-PE, IgD-PB, CD21/35 (7E9)-PE, CD138-PE, CD38-PE-Cy7, CD31-A647, CD157-PE, Streptavidin-PE/Cy7; from eBioscience: CD95 (APO-1/Fas)-PE (clone 15A7) and viability dye Fixable live/dead stain Efluor780; from ThermoFisher Scientific: Rabbit-anti-Goat-A488, Hoechst 33342, DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), Phalloidin-A568; from Southern Biotech: AP-Goat-anti-Mouse IgG, AP-Goat-anti-Mouse IgG2a, AP-Goat-anti-Mouse IgG2c; from Rockland Immunochemicals: Rabbit polyclonal anti-B-Phycoerythrin; from DAKO: Rabbit polyconal anti-Mouse Immunoglobulins-biotin; from Perkin-Elmer: Europium-labeled streptavidin. In-house generated anti-idiotypic Ab, clone 9D11, conjugated to Alexa Fluor 647 or Alexa Fluor 568; in-house generated Rabbit polyclonal anti-C3b conjugated to Alexa Fluor 633.

Degn S.E., van der Poel C.E., Firl D.J., Ayoglu B., Al Qureshah F.A., Bajic G., Mesin L., Reynaud C.A., Weill J.C., Utz P.J., Victora G.D, & Carroll M.C. (2017). Clonal Evolution of Autoreactive Germinal Centers. Cell, 170(5), 913-926.e19.

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Aortic Immune Cell Profiling by FACS

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For fluorescence‐activated cell sorting analysis of aortic tissue, each aorta was excised above the branching point of the right renal artery along with a length of 10 mm. Tissue was digested with 400 U/mL of collagenase II (#LS004176; Worthington Labs, Lakewood, NJ) and 0.75 U/mL of elastase (#LS002274; Worthington Labs) at 37°C for 90 minutes. After enzymatic isolation, cells were stained with CD11b‐PE‐Cy7 (#25‐0112‐81; eBioscience, San Diego, CA), CD45.2‐FITC (#11‐0454‐81; eBioscience), Ly6C‐PE (#1280019; BioLegend, San Diego, CA), Ly6G‐APC (#127613; BioLegend), and 7‐aminoactinomycin D (#559925; BD Biosciences, San Jose, CA). Control samples with single‐color staining were used for determining the staining conditions and fluorescence signal compensation. Samples were subjected to fluorescence‐activated cell sorting analysis using the FACSCanto II (BD Biosciences), followed by data visualization using FlowJo software (Tree Star).

Ohno‐Urabe S., Aoki H., Nishihara M., Furusho A., Hirakata S., Nishida N., Ito S., Hayashi M., Yasukawa H., Imaizumi T., Akashi H., Tanaka H, & Fukumoto Y. (2018). Role of Macrophage Socs3 in the Pathogenesis of Aortic Dissection. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(2), e007389.

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Fetal Hematopoietic Stem Cell Transplantation

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Fetal HSCs (Lin-/Sca-1+/c-Kit+/Flk2-/CD150+/CD48-) were isolated from the fetal livers of E14.5 Mcm3^+/+, Mcm3^+/Lox or Mcm3^Lox/Lox CD45.2 C57Bl/6 donor mice, and transplanted into lethally irradiated CD45.1 C57Bl/6 primary recipients (50 fetal HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells. 4 months post-transplantation, the primary recipients were sacrificed to assess the percentage of donor-derived chimerism in the bone marrow (BM), spleen, peripheral blood and HSC compartment and to re-isolate donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs53 (link). Donor chimerism was analyzed using CD45.2-FITC, (eBioscience 12-0453-83; 1:50), B220-APC-e780 (47-0452-82; 1:800), Gr-1-PB (1:400), Mac-1-PE-Cy7 (1:3200), CD3-e660 (eBioscience, 50-0032-82; 1:400) and Ter-119-PE-Cy5 (eBioscience 15-5921-83; 1:800) antibodies. Re-isolated CD45.2⁺ donor-derived adult MCM3^+/+ or MCM3^Lox/Lox HSCs were transplanted into lethally irradiated CD45.1 secondary recipients (500 HSCs per mouse; 5 mice per group) together with 300,000 Sca-1-depleted CD45.1 helper BM cells.

Alvarez S., Díaz M., Flach J., Rodriguez-Acebes S., López-Contreras A.J., Martínez D., Cañamero M., Fernández-Capetillo O., Isern J., Passegué E, & Méndez J. (2015). Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality. Nature Communications, 6, 8548.

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Multicolor Flow Cytometry Analysis

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For analysis of cell surface markers, cells were stained in PBS containing 2% heat-inactivated FBS with 2 mM EDTA supplementation, unless noted otherwise. Antibodies used were as follows: CD4-PerCP/Cy5.5, CD8α-APC, and CD127-APC (BioLegend); CD4-PE and CD44-PE (BD); CD62L-FITC, CD25-PE, CD45.2-FITC, and CD45.1-PE (eBioscience). Foxp3-APC and eFluor780 (eBioscience), Annexin V-FITC, and 7-AAD (BD) were stained according to the manufacturer’s instructions. Cells were analyzed with FACSCalibur or FACSCanto II flow cytometry (BD) using FlowJo software (Tree Star).

Matsushita M., Freigang S., Schneider C., Conrad M., Bornkamm G.W, & Kopf M. (2015). T cell lipid peroxidation induces ferroptosis and prevents immunity to infection. The Journal of Experimental Medicine, 212(4), 555-568.

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