Cd45ro pe cf594

Manufactured by BD

Sourced in Belgium, United States

CD45RO PE-CF594 is a fluorescently-labeled antibody designed for the detection and analysis of CD45RO-expressing cells using flow cytometry. CD45RO is an isoform of the CD45 protein expressed on the surface of memory T cells. The PE-CF594 fluorescent dye is conjugated to the antibody, allowing for its detection and quantification.

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8 protocols using cd45ro pe cf594

Multiparametric Immune Cell Phenotyping

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Antibodies for surface staining included CCR7 APC-Cy7 (clone G043H7; Biolegend), CCR7 APC-eFluor780 (clone 3D12; eBioscience), CD4 PE-Cy5.5 (clone S3.5; Invitrogen), CD8 BV711 (clone RPA-T8; Biolegend), CD8 Qdot 605 (clone 3B5; Invitrogen), CD14 BV510 (clone M5E2; Biolegend), CD14 Pacific Blue (clone M5E2; custom), CD14 PE-Cy5 (clone 61D3; Abcam), CD14 PE-Cy7 (clone HCD14; Biolegend), CD16 Pacific Blue (clone 3G8; custom), CD16 PE-Cy5 (clone 3G8; Biolegend), CD16 PE-Cy7 (clone 3G8; Biolegend), CD19 BV510 (clone HIB19; Biolegend), CD19 Pacific Blue (clone HIB19; custom), CD19 PE-Cy5 (clone HIB19; Biolegend), CD19 PE-Cy7 (clone HIB19; Invitrogen), CD45RO ECD (clone UCHL1; Beckman Coulter), CD45RO PE-CF594 (clone UCHL1; BD Biosciences), CD107a PE-Cy5 (clone eBioH4A3; eBioscience), CD107a PE-Cy7 (clone H4A3; Biolegend), and HLA-DR Pacific Blue (clone LN3; Invitrogen). Antibodies for intracellular staining included CD3 BV570 (clone UCHT1; Biolegend), CD3 BV650 (clone OKT3; Biolegend), CD3 Qdot 585 (clone OKT3; custom), CD3 Qdot 650 (clone S4.1; Invitrogen), Eomes Alexa 647 (WD1928; eBioscience), Eomes eFluor 660 (WD1928; eBioscience), IFN-γ Alexa 700 (clone B27; Invitrogen), Perforin BV421 (clone B-D48, Biolegend), Perforin Pacific Blue (clone B-D48; custom), Perforin PE (clone B-D48, Cell Sciences), T-bet FITC (clone 4B10; Biolegend), and T-bet PE (clone 4B10; eBioscience).

Demers K.R., Makedonas G., Buggert M., Eller M.A., Ratcliffe S.J., Goonetilleke N., Li C.K., Eller L.A., Rono K., Maganga L., Nitayaphan S., Kibuuka H., Routy J.P., Slifka M.K., Haynes B.F., McMichael A.J., Bernard N.F., Robb M.L, & Betts M.R. (2016). Temporal Dynamics of CD8+ T Cell Effector Responses during Primary HIV Infection. PLoS Pathogens, 12(8), e1005805.

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Multiparameter Flow Cytometry Profiling of Activated cTfh

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CD3-Alexa Fluor 700, CD4-BV605, CD8-APCy7, CD38-APC, TIGIT-PE-Cy7, CD45RO-PECF594, CCR7-BV421, PD1-PE, ICOS-PcPCy5.5 and CXCR5-AF488 (BD Biosciences).
Cells were washed twice and run/sorted on a BD FACS Aria III. Samples from 12 study participants were selected based on the magnitude of their cTfh response from d0 to d7 to undergo bulk sorting, with samples from four of these participants also undergoing single cell sorting. Sorted cells had 100U/mL of RNaseOUT added prior to sorting. Bulk sorted cells were centrifuged with the supernatant removed prior to freezing at -80°C. Data were analysed with FlowJo 10.1 (TreeStar). Activated and resting cTfh cells (CD4⁺CXCR5⁺PD-1⁺) were defined as CD38⁺ICOS⁺ and CD38^-ICOS^-, respectively (19 (link), 22 (link)). Activated plasmablasts were defined as CD19⁺CD27⁺CD38⁺. An example of flow gating for activation and sorting is depicted in Supplementary Figure 8.

Currenti J., Simmons J., Oakes J., Gaudieri S., Warren C.M., Gangula R., Alves E., Ram R., Leary S., Armitage J.D., Smith R.M., Chopra A., Halasa N.B., Pilkinton M.A, & Kalams S.A. (2023). Tracking of activated cTfh cells following sequential influenza vaccinations reveals transcriptional profile of clonotypes driving a vaccine-induced immune response. Frontiers in Immunology, 14, 1133781.

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Profiling Memory CD4+ T Cells

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Frozen PBMCs obtained from the patient and two healthy donors were thawed when cells (10⁶) were either unstimulated or stimulated with PMA (20ng/mL; Sigma) and ionomycin (1μM; Sigma) in the presence of brefeldin A (10μg/mL; Sigma) at 37°C overnight. For detection of intracellular cytokines, cells were stained with Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen) as well as for extracellular CD3 (eVolve605), CD8 APC-eFluor780 (eBioscience), CD45RO PE-CF594 (BD Biosciences), and CD27 BV421 (BioLegend) for 30 min. Cells were fixed (paraformaldehyde 4%), permeabilized, and stained for intracellular antigens with CD4 PE-Cy7 (BD Biosciences), IL-17A PE, IFN-γ FITC, and TNF-α APC (eBioscience). Data were collected using an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software (Treestar) by gating on lymphocytes (forward & side scatter) and on viable CD3+CD4+CD8-CD45RO+ CD27− cells.

Sampaio E.P., Ding L., Rose S.R., Cruz P., Hsu A.P., Kashyap A., Rosen L.B., Smelkinson M., Tavella T.A., Ferre E.M., Wierman M.K., Zerbe C.S., Lionakis M.S, & Holland S.M. (2017). NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION. The Journal of allergy and clinical immunology, 141(5), 1844-1853.e2.

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Comprehensive B and T Cell Immunophenotyping

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PB was collected in heparin-coated tubes (Venosafe plastic tubes, Terumo Europe N.V., Leuven, Belgium) and PB mononuclear cells (PBMC) were isolated using high density centrifugation (Lympholyte; Cedarlane Laboratories, SanBio B.V., Uden, the Netherlands). PBMC (0.5×10⁶ cells) were stained using anti-human CD19 PerCP-Cy5.5 and CD4 APC to discriminate between B and T cells, respectively (BD Biosciences, Erembodegem, Belgium). B cell subpopulations and surface molecules were defined using following anti-human antibodies: IgD APC-Cy7, CD27 PE-Cy7, HLA-DR/DP/DQ (major histocompatibility complex (MHC)-II) FITC, CD80 PE and CD86 PE-CF594 (all from BD Biosciences, Erembodegem, Belgium). Following anti-human monoclonal antibodies were used for T cell analysis: CD45RA APC-H7, CD45RO PE-CF594, CXCR5 Alexa Fluor 488 and PD-1 PE-Cy7 (all from BD Biosciences, Erembodegem, Belgium), CD25 PerCP-Cy5.5 and CD127 PE (eBioscience, San Diego, USA). Following isotype controls were used: mouse IgG1 PErCP-Cy5.5, IgG1 PE, IgG1 PE-Cy7, IgG2aκ PE-CF594, IgG2bκ APC-H7, IgG1 APC, IgG2aκ FITC, IgG1κ PE-CF594, IgG1 PE-Cy7, IgG2aκ APC-H7 and rat IgG2b Alexa Fluor 488 (all from BD Biosciences, Erembodegem, Belgium). All flow cytometric analyses were performed on a FACSAriaII flow cytometer and analyzed with FACS Diva software (BD Biosciences).

Claes N., Dhaeze T., Fraussen J., Broux B., Van Wijmeersch B., Stinissen P., Hupperts R., Hellings N, & Somers V. (2014). Compositional Changes of B and T Cell Subtypes during Fingolimod Treatment in Multiple Sclerosis Patients: A 12-Month Follow-Up Study. PLoS ONE, 9(10), e111115.

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Comprehensive Immune Cell Profiling

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All antibodies were used at the concentration recommended by the manufacturer or pre-titrated for optimal staining. The following antibodies were used in this study; anti-CD3 APC-eFluor 780 (UCHT1), -CD4 APC (OKT4), -CD161 PE-Cy7 (HP-3G10), -IFNγ eFluor450 or PE (45.B3), -IL-17A eFluor 488 (eBio64DEC17), -IL-22 PE (22URT1), -IL-21 PE (eBio3A3-N2), -IL-17F PE (SHLR17), -T-bet PE (eBio4B10), -RORγt PE (AFKJS-9) (eBioscience), anti-CD3 Brilliant Violet 510 (OKT 3), -CD4 PE-Cy7 (OKT4), -CD8 Brilliant Violet 510 (RPA-T8), -CD25 PerCP-Cy5.5 (BC96), -CCR6 PE or Alexa Fluor (AF) 647 (G034E3), -CCR7 AF488 (G043H7), -CXCR3 AF647 (G025H7), anti-CD45RA PE-CF594 (HI100), -CD45RO PE-CF594 (UCHL1), -GM-CSF PE (BVD2-21C11) (BD Pharmingen), and anti-CD4 PE-Vio770 (VIT 4) (Miltenyi Biotec).

Restorick S.M., Durant L., Kalra S., Hassan-Smith G., Rathbone E., Douglas M.R, & Curnow S.J. (2017). CCR6+ Th cells in the cerebrospinal fluid of persons with multiple sclerosis are dominated by pathogenic non-classic Th1 cells and GM-CSF-only-secreting Th cells. Brain, Behavior, and Immunity, 64, 71-79.

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Comprehensive T Cell Phenotyping

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A multi-color flow cytometry panel was designed to assess T cell phenotype markers. At select time points during expansion, T cells were stained using an antibody cocktail (CD4-BUV395, CD8-APC-H7, CD25-PE-Cy7, CD27-BUV737, CD45RA-BV650, CD45RO-PE-CF594, CD69-BV786, CD197/CCR7-PE, and CD279/PD1-BV421; BD Biosciences) diluted in staining solution (1:1 mixture of Brilliant Stain Buffer Plus [BD Biosciences, #566385] and PBS with 1% FBS, 10 mM HEPES, and 2 mM EDTA) for 30 min at room temperature, centrifuged, re-suspended in fixation solution (1% formaldehyde in PBS), and immediately analyzed.

Bloemberg D., Nguyen T., MacLean S., Zafer A., Gadoury C., Gurnani K., Chattopadhyay A., Ash J., Lippens J., Harcus D., Pagé M., Fortin A., Pon R.A., Gilbert R., Marcil A., Weeratna R.D, & McComb S. (2020). A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells. Molecular Therapy. Methods & Clinical Development, 16, 238-254.

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Multiparametric Flow Cytometry of PBMCs

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Cryopreserved PBMCs were thawed, washed, and stained with Live/Dead Aqua Blue (Life Technologies), followed by surface staining with antihuman CD3 BV605, anti-human CD4 BV650, anti-human CD8 BV711, anti-human CD27 PerCP-eF710, CD45RO PE-CF594 (BD Biosciences), anti-human CD98 PE (BD Biosciences), anti-human CD71 APC, anti-human PD-1 PE-Cy7, and anti-human T-Bet BV421 (all from BioLegend unless otherwise noted). Anti-human CD14, anti-human CD16, and anti-human CD19 all tagged with V500 (BD Biosciences) were added in dump. For median fluorescence index determination, manual target channeling was performed with 1× beads (SpiroTech). Samples were run on modified 5 laser BD LSR Fortessa X-20. Data were analyzed on FlowJo (FlowJo).

Rahman A.N., Liu J., Mujib S., Kidane S., Ali A., Szep S., Han C., Bonner P., Parsons M., Benko E., Kovacs C., Yue F.Y, & Ostrowski M. (2021). Elevated glycolysis imparts functional ability to CD8+ T cells in HIV infection. Life Science Alliance, 4(11), e202101081.

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Multiparametric Flow Cytometry of Immune Cells

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For cell surface markers, cells were stained in PBS containing 2% fetal bovine serum for 15 minutes, unless mentioned otherwise. For intracellular cytokine staining, cells were stimulated with phorbol 12-myristate acetate (PMA) (50 ng/ mL) and ionomycin (1 lg/mL), or with different antigens, the last 8 hours with 10 lg/mL Brefeldin A and/or 2 lmol/mL monensin, at 378C and 5% CO 2 as mentioned above, and then fixed and stained according to manufacturer's protocols. Antibodies used were as follows: CD3-Alexa Fluor 700, CD4-PerCP/Cy5.5A, IFN-c Alexa Fluor 488, IL-17-PerCP/Cy5.5A, CCR7-APCeFluor780 (eBioscience, San Diego, CA, USA), CD8-V500A (Tonbo, San Diego, CA, USA), TNF-a-Alexa Fluor 700(BD), CD45RA-PECy7, CD45RO-PECF594, FAS-PECF594 (BD, San Diego, CA, USA), FasL-PE (Invitrogen), AnnexinV-APC (Immuno Tools, GmbH, Germany). Cells were acquired and analyzed by BD LSR Fortessa II (BD), using FlowJo (Ashland, OR, USA) three-star or FACSDIVA 6 software (BD Biosciences, San Jose, CA, USA).

Tagirasa R., Parmar S., Barik M.R., Devadas S, & Basu S. (2017). Autoreactive T Cells in Immunopathogenesis of TB-Associated Uveitis. Investigative ophthalmology & visual science, 58(13).

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