Cd45ro microbeads

Manufactured by Miltenyi Biotec

Sourced in Germany

CD45RO microbeads are an immunomagnetic labeling reagent used for the isolation and enrichment of CD45RO+ cells from biological samples. CD45RO is a cell surface marker expressed on memory T cells. The microbeads are coated with antibodies specific to the CD45RO antigen, allowing for the selective isolation of CD45RO+ cells.

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14 protocols using cd45ro microbeads

Isolation of Naïve CD4 T Cells from Human Blood

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Peripheral blood and leukapheresis samples were obtained from 85 blood donors in the age ranges of 20–35 and 60–85 years. All of these individuals were healthy, meeting the Stanford Blood bank criteria for blood donation. Samples had been de‐identified except for age range. For the experiments in Figure 1, we collected blood from additional seven female and six male young and old volunteers, who did not have an acute or a poorly controlled chronic disease and who were not frail. The study was approved by the Stanford Institutional Review Board, and all participants gave informed consent. CD4 T cells were isolated with Human CD4 T Cell Enrichment Cocktail (STEMCELL Technologies). Naïve CD4 T cells were further purified by negative selection with CD45RO microbeads by magnetic‐activated cell sorting (Miltenyi Biotec). Purity of CD3⁺CD4⁺CD45RA⁺CCR7⁺ cells was confirmed to be higher than 95% by flow analysis (Figure S6).

Hu B., Li G., Ye Z., Gustafson C.E., Tian L., Weyand C.M, & Goronzy J.J. (2019). Transcription factor networks in aged naïve CD4 T cells bias lineage differentiation. Aging Cell, 18(4), e12957.

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Activation of Naïve CD4+ T Cells

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CD4+ T cells were obtained by apheresis from de-identified healthy blood donors after informed consent by the University of Pennsylvania Human Immunology Core. Samples were collected from three donors, Donor 1 (age: 46, sex: male), Donor 2 (age: 26, sex: female), and Donor 3 (age: 32, sex: male). Naïve CD4+ T cells were negatively enriched for by utilizing CD45RO microbeads (Miltenyi: 130-046-001). 5×10⁶ naïve CD4+ T cells were stimulated in complete RPMI supplemented with 10 IU of IL-2 (Miltenyi: 130-097-742), with either soluble 2.5 µg/mL anti-CD28 (BD: 348040) or 2.5 µg/mL bound anti-CD3 (BD: 555336), or with both soluble anti-CD28 and bound anti-CD3. Cells were harvested after 8 and 48 hr of culture. Stimulation efficiency and consistency between human donors was measured by CD69 expression (Biolegend: 310905), analyzed by flow cytometry on the BD LSRII equipment maintained by the Flow Cytometry core at the University of Pennsylvania. Growth and transfection of Jurkat cells was as described previously (Lynch and Weiss, 2000 (link)). Jurkat cells were validated by RNA-sequencing and negative for mycoplasm.

Blake D., Radens C.M., Ferretti M.B., Gazzara M.R, & Lynch K.W. (2022). Alternative splicing of apoptosis genes promotes human T cell survival. eLife, 11, e80953.

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Isolation and Activation of Naive T Cells

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PBMCs from adult leukocyte reduction filters [21 (link),22 (link)], CB samples, and peripheral blood were isolated by Ficoll^™ gradient centrifugation (Biochrom, Berlin, Germany). CD4⁺CD45RA⁺CD31⁺ naive T cells were enriched, using a CD4⁺ T cell Isolation Kit II and AutoMACS (magnetic-assisted cell sorting) (Miltenyi Biotec, Bergisch-Gladbach, Germany) followed by negative selection by CD45RO Micro Beads and positive selection by anti-CD31 Micro Beads (Miltenyi Biotec) The purified CD45RA⁺ fractions contained 95–97% CD45RA⁺ cells, and the purified CD31⁺ fractions contained 87–99% CD31⁺ T cells. The cells were cultivated at 37°C in RPMI 1640 medium (Biochrom, Berlin, Germany), supplemented with penicillin-streptomycin and with 10% human AB-plasma. A total of 2×10⁶ T cells/ml were loaded with anti-CD3 Ab (0.5 μg/ml) plus anti-CD28 Ab (0.5 μg/ml) for two minutes, then stimulation was performed by cross-linking the Abs with 10 μg/ml goat anti-mouse IgG (GAMIg) (Invitrogen, Darmstadt, Germany).

Schmiedeberg K., Krause H., Röhl F.W., Hartig R., Jorch G, & Brunner-Weinzierl M.C. (2016). T Cells of Infants Are Mature, but Hyporeactive Due to Limited Ca2+ Influx. PLoS ONE, 11(11), e0166633.

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Isolation and Separation of T-Cell Subsets

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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation as previously described.2 (link) CD4⁺ and CD8⁺ T cells were obtained using negative selection MACS-kits according to manufacturer's instructions (CD4⁺ T-Cell Isolation Kit and CD8⁺ T-Cell Isolation Kit; Miltenyi, Bergisch Gladbach, Germany). In a second step, CD45RA⁺ and CD45RO⁺ T cells were obtained via positive selection using CD45RA or CD45RO Microbeads (Miltenyi).

Fessler J., Raicht A., Husic R., Ficjan A., Duftner C., Schwinger W., Dejaco C, & Schirmer M. (2015). Premature senescence of T-cell subsets in axial spondyloarthritis. Annals of the Rheumatic Diseases, 75(4), 748-754.

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Isolation and Characterization of CD4+ T Cell Subsets

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Peripheral blood samples were collected into EDTA vacutainer tubes (Greiner Bio-One). PBMCs were isolated from the blood of healthy volunteers, RA, and PsA patients by density gradient centrifugation over Histopaque-1077 (Sigma-Aldrich). For quantitative real-time PCR analysis, the CD4⁺ T cells were negatively isolated from PBMCs with a magnetic-activated cell sorting CD4⁺ T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. The CD45RO⁻ (negative fractions) and CD45RO⁺ (positive fractions) cells were separated also with a CD45RO Microbeads (Miltenyi Biotec). For flow cytometric analysis of chemokine receptors, the CD4⁺CD45RO⁻ cells were negatively isolated from PBMCs with naive CD4⁺ T Cell isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of the separated cells was at least 95% in all cases, as determined by flow cytometry.

Baricza E., Marton N., Királyhidi P., Kovács O.T., Kovácsné Székely I., Lajkó E., Kőhidai L., Rojkovich B., Érsek B., Buzás E.I, & Nagy G. (2018). Distinct In Vitro T-Helper 17 Differentiation Capacity of Peripheral Naive T Cells in Rheumatoid and Psoriatic Arthritis. Frontiers in Immunology, 9, 606.

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CAR T-cell Manufacturing from Naive and Memory T-cell Subsets

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Healthy donor T-cells were isolated from PBMCs using the Pan T-cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec). To manufacture CAR T-cells from naïve, central memory, effector memory, and effector T-cells, each T-cell subset was magnetically isolated using the REAlease CD62 microbead kit (Miltenyi Biotech) and CD45RO microbeads (Miltenyi Biotech). T-cells were maintained in OpTmizer CTS SFM media (Gibco) supplemented with 5% human AB serum and 100U/mL human IL-2. On day zero, CD3/CD28 Dynabeads CTS^™ (Thermo Fisher Scientific) were added to T-cells at a bead to cell ratio of 3:1. After 24-hours of bead-based activation, lentiviral vector encoding an anti-CD19, anti-Mesothelin or anti-PSMA CAR was added to the culture at a multiplicity of infection (MOI) of two. Media was added to cultures every two days and CAR T-cells were maintained at a constant concentration of 5 × 10⁵ cells/mL, followed by harvest and cryopreservation on day nine of the CAR T-cell expansion process.

Jung I.Y., Bartoszek R.L., Rech A.J., Collins S.M., Ooi S.K., Williams E.F., Hopkins C.R., Narayan V., Haas N.B., Frey N.V., Hexner E.O., Siegel D.L., Plesa G., Porter D.L., Cantu A., Everett J.K., Guedan S., Berger S.L., Bushman F.D., Herbst F, & Fraietta J.A. (2023). Type I Interferon Signaling via the EGR2 Transcriptional Regulator Potentiates CAR T cell-intrinsic Dysfunction. Cancer discovery, 13(7), 1636-1655.

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Isolation of Naive and Memory CD4+ T Cells

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Naïve and memory CD4+ T cells were isolated using a RosetteSep Human CD4+ T cell Enrichment Cocktail (Stemcell Technologies) followed by CD45RO MicroBeads (Miltenyi Biotech), achieving consistent median purities of 82.4 and 78.7%, respectively. For whole CD4+ T cell experiments cells were isolated using monocyte depletion by immuno-rosetting followed by automated magnetic bead-based positive selection (median 99.1% purity; Stemcell Technologies). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation on Lymphoprep (Axis-Shield Diagnostics, Dundee, UK).

Ridgley L.A., Anderson A.E., Maney N.J., Naamane N., Skelton A.J., Lawson C.A., Emery P., Isaacs J.D., Carmody R.J, & Pratt A.G. (2019). IL-6 Mediated Transcriptional Programming of Naïve CD4+ T Cells in Early Rheumatoid Arthritis Drives Dysregulated Effector Function. Frontiers in Immunology, 10, 1535.

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Isolation and Differentiation of Immune Cells

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Buffy coats from the healthy donors were processed to purify peripheral blood mononuclear cells (PBMCs). Ethics committee approval for the use of such material (Institut National de la Santé et de la Recherche-EFS ethical committee convention 15/EFS/012) was obtained and experiments were performed in accordance with the approved guidelines of INSERM. The CD4⁺ T cell isolation kit-II (Miltenyi Biotec, Paris, France) was used to isolate untouched total CD4⁺ T cells by negative selection. Subsequently, CD45RA⁺ and CD45RO⁺ CD4⁺ T cells were separated by using CD45RO microbeads (Miltenyi Biotec). Furthermore, CD25⁺ cells were depleted from the CD45RA⁺ fraction by using CD25 microbeads (Miltenyi Biotec) to obtain CD4⁺CD25⁻CD45RO⁻ naïve T cells. The purity of all subpopulations was more than 96%.
Monocytes were isolated from PBMC by using CD14 microbeads (Miltenyi Biotec) and were cultured for 5 days with GM-CSF (1,000 IU/million cells) and IL-4 (500 IU/million cells) (both from Miltenyi Biotec) for the differentiation into DCs (17 (link)).

Saha C., Das M., Patil V., Stephen-Victor E., Sharma M., Wymann S., Jordi M., Vonarburg C., Kaveri S.V, & Bayry J. (2017). Monomeric Immunoglobulin A from Plasma Inhibits Human Th17 Responses In Vitro Independent of FcαRI and DC-SIGN. Frontiers in Immunology, 8, 275.

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Generating CD19- and GD2-targeting CAR-T cells

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Peripheral blood mononuclear cells (PBMCs) derived from healthy donors (Cellular Technology Limited, Cleveland, OH) were stimulated with mitomycin C-treated K562 cells (JCRB, Osaka, Japan) expressing anti-CD3 mAb (clone OKT3)-derived single-chain variable fragment (scFv) and CD80 on the cell surface at an effector to target ratio of 7:1 (4 (link)). Stimulated T cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and recombinant IL-2 (100 IU/ml, Nipro, Osaka, Japan). Unstimulated T cells were maintained in the presence of recombinant IL-7 (10 ng/ml, PeproTech, Waltham, MA). When indicated, naïve T cells were enriched by magnetically isolating CD45RO⁻ cells using CD45RO MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany).
To generate CAR-T cells, T cells were retrovirally transduced with CD19- or GD2-targeting CAR genes two days after stimulation. We used PG13 packaging cells for retrovirus production. The CAR constructs consisted of a single-chain variable fragment (scFv) derived from the clone FMC63 (targeting CD19) (24 (link)) or 14g2a with E101K high-affinity mutation (targeting GD2) (25 (link)) linked to CD28 and CD3z signaling domains.

Ito Y., Inoue S., Nakashima T., Zhang H., Li Y., Kasuya H., Matsukawa T., Wu Z., Yoshikawa T., Kataoka M., Ishikawa T, & Kagoya Y. (2023). Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells. Nucleic Acids Research, 52(1), 141-153.

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Isolation and Activation of Naïve CD4+ T Cells

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CD4⁺ T cells were obtained by apheresis from de-identified healthy blood donors after informed consent by the University of Pennsylvania Human Immunology Core. Samples were collected from three donors, Donor 1 (age: 46, sex: male), Donor 2 (age: 26, sex: female), and Donor 3 (age: 32, sex: male). Naïve CD4⁺ T cells were negatively enriched for by utilizing CD45RO microbeads (Miltenyi: 130-046-001). 5 × 10⁶ naïve CD4⁺ T cells were stimulated in complete RPMI supplemented with 10 IU of IL-2 (Miltenyi: 130-097-742), with either soluble 2.5 μg/mL anti-CD28 (BD: 348040), or 2.5 μg/mL bound anti-CD3 (BD: 555336), or with both soluble anti-CD28 and bound anti-CD3. Cells were harvested after 8 h and 48 h of culture. Validation of stimulation is described elsewhere^{18 (link)}.

Blake D., Gazzara M.R., Breuer I., Ferretti M, & Lynch K.W. (2024). Alternative 3′UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner. Scientific Reports, 14, 10987.

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