Cd45ro uchl1, by BD - Product details

1

Multiparametric Flow Cytometry for T Cell Phenotyping

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T cell phenotype was assessed by flow cytometry (LSRFortessa, BD) using fluorochrome-conjugated antibodies specific for CD3 (clone UCHT1; BD), CD4 (SK3; BD), CD8 (SK1; BD), CD45RO (UCHL1; BD), CX3CR1 (2A9-1; BioLegend), CD57 (HNK-1; BioLegend), CD27 (M-T271; BD), CD28 (CD28.2; BioLegend), CD69 (FN50; BD), and CCR7 (3D12; BD). Viable cells were gated using Live/Dead Aqua viability dye (Invitrogen). For induction and detection of intracellular cytokines, cells were cultured overnight with 20ng/ml recombinant human IL-15 (247-ILB; R&D Systems) or medium control (RPMI 1640 [Gibco], supplemented with 10% fetal bovine serum [Gemini Bio-Products], 1% L-glutamine [Gibco], and 1% penicillin/streptomycin [Gibco]), then treated with brefeldin A (GolgiPlug, BD) for 6h prior to harvest. After Live/Dead and surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD) for 20 min on ice and stained for 40 minutes on ice with anti-IFNγ (B27, BD) and anti-TNF (MAb11, BD). For intracellular accumulation of cytolytic molecules, cells were treated with IL-15-supplemented or control medium for 48 hours, then harvested, stained with Live/Dead and surface antibodies, treated with Cytofix/Cytoperm, stained with anti-granzyme B (GB11; BD) and anti-perforin (B-D48; BioLegend).

Panigrahi S., Chen B., Fang M., Potashnikova D., Komissarov A.A., Lebedeva A., Michaelson G.M., Wyrick J.M., Morris S.R., Sieg S.F., Paiardini M., Villinger F.J., Harth K., Kashyap V.S., Cameron M.J., Cameron C.M., Vasilieva E., Margolis L., Younes S.A., Funderburg N.T., Zidar D.A., Lederman M.M, & Freeman M.L. (2020). CX3CL1 and IL-15 Promote CD8 T cell chemoattraction in HIV and in atherosclerosis. PLoS Pathogens, 16(9), e1008885.

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2

Comprehensive Immunophenotyping of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll step gradient and stained for cell surface markers with the following fluorochrome-conjugated antibodies: CD38 (HIT-2, BD), IgD (IA6-2, BD), IgM (MHM-88, BioLegend), CD10 (HI10a, BD), CD19 (SJ25C1, BD), CD21 (B-ly4, BD), CD27 (0323, eBiosience), CD138 (B-B4, AbD SeroTec), IgG (SouthernBioTech #2043-09), IgA (G20-359, BD), CD3 (SK7, BD), CD28 (CD28.2, BD), FOXP3 (PCH101, eBioscience), CD8 (SK1, BD), CD4 (RPA-T4, eBioscience), γδ-TCR (B1.1, eBioscience), αβ-TCR (IP26, eBioscience), CD45RO (UCHL1, BD), CD45RA (HI100, BD), CD57 (NK-1, BD), CD31 (WM59, eBioscience), CD62L (DREG-56, BD), CD152/CTLA4 (BNI3, BD). Samples were acquired on a FACS-Canto II flow cytometer (BD) or on a Gallios flow cytometer (Beckman Coulter, Miami, FL) and analyzed using FlowJo version 7.6.5 analysis software (Treestar, Ashland, OR).

Schubert D., Bode C., Kenefeck R., Hou T.Z., Wing J.B., Kennedy A., Bulashevska A., Petersen B.S., Schäffer A.A., Grüning B.A., Unger S., Frede N., Baumann U., Witte T., Schmidt R.E., Dueckers G., Niehues T., Seneviratne S., Kanariou M., Speckmann C., Ehl S., Rensing-Ehl A., Warnatz K., Rakhmanov M., Thimme R., Hasselblatt P., Emmerich F., Cathomen T., Backofen R., Fisch P., Seidl M., May A., Schmitt-Graeff A., Ikemizu S., Salzer U., Franke A., Sakaguchi S., Walker L.S., Sansom D.M, & Grimbacher B. (2014). Autosomal-dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nature medicine, 20(12), 1410-1416.

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3

PBMC Signaling Pathway Analysis

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PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 10⁶/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO₂ incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.

Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.

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4

Comprehensive Immunophenotyping of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll step gradient and stained for cell surface markers with the following fluorochrome-conjugated antibodies: CD38 (HIT-2, BD), IgD (IA6-2, BD), IgM (MHM-88, BioLegend), CD10 (HI10a, BD), CD19 (SJ25C1, BD), CD21 (B-ly4, BD), CD27 (0323, eBiosience), CD138 (B-B4, AbD SeroTec), IgG (SouthernBioTech #2043-09), IgA (G20-359, BD), CD3 (SK7, BD), CD28 (CD28.2, BD), FOXP3 (PCH101, eBioscience), CD8 (SK1, BD), CD4 (RPA-T4, eBioscience), γδ-TCR (B1.1, eBioscience), αβ-TCR (IP26, eBioscience), CD45RO (UCHL1, BD), CD45RA (HI100, BD), CD57 (NK-1, BD), CD31 (WM59, eBioscience), CD62L (DREG-56, BD), CD152/CTLA4 (BNI3, BD). Samples were acquired on a FACS-Canto II flow cytometer (BD) or on a Gallios flow cytometer (Beckman Coulter, Miami, FL) and analyzed using FlowJo version 7.6.5 analysis software (Treestar, Ashland, OR).

Schubert D., Bode C., Kenefeck R., Hou T.Z., Wing J.B., Kennedy A., Bulashevska A., Petersen B.S., Schäffer A.A., Grüning B.A., Unger S., Frede N., Baumann U., Witte T., Schmidt R.E., Dueckers G., Niehues T., Seneviratne S., Kanariou M., Speckmann C., Ehl S., Rensing-Ehl A., Warnatz K., Rakhmanov M., Thimme R., Hasselblatt P., Emmerich F., Cathomen T., Backofen R., Fisch P., Seidl M., May A., Schmitt-Graeff A., Ikemizu S., Salzer U., Franke A., Sakaguchi S., Walker L.S., Sansom D.M, & Grimbacher B. (2014). Autosomal-dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nature medicine, 20(12), 1410-1416.

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5

T-cell Cytotoxicity and Proliferation Assays

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T-cell assays for activity, proliferation and cytotoxicity have been described in detail elsewhere [30 (link)]. Briefly, in coculture experiments, T cells were incubated with irradiated U87vIII target cells at an E:T of 1:1 for time periods as described. Cell-free supernatants from cells were also analyzed for cytokine expression using a Luminex array (Luminex Corp, FLEXMAP 3D) according to manufacturers instructions. Expression of surface markers were either taken at baseline or after a period of coculture, and then subjected to flow cytometric analysis. Antigens were stained for using the following antibody clones for flow cytometry where indicated: CCR7 (3E12, BD Bioscience); CD45RO (UCHL1, BD Biosciences), PD-1 (EH12287, Biolegend). For proliferation assays, cells were stimulated with irradiated target cells at an E:T of 1:1. Cells were counted every 7 days and plated again with stimulation at 7 day intervals. In experiments when real-time cytotoxicity was measured against U87vIII, cell index was recorded as a measure of cell impedance using the xCELLigence RTCA SP instrument (ACEA Biosciences, Inc.) according to manufacturer instructions. Percent specific lysis may be calculated from these data using the following equation: % = ((cell index of UTDs - cell index of CAR T cells) / cell index of UTDs) × 100.

Choi B.D., Yu X., Castano A.P., Darr H., Henderson D.B., Bouffard A.A., Larson R.C., Scarfò I., Bailey S.R., Gerhard G.M., Frigault M.J., Leick M.B., Schmidts A., Sagert J.G., Curry W.T., Carter B.S, & Maus M.V. (2019). CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma. Journal for Immunotherapy of Cancer, 7, 304.

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6

PBMC Signaling Pathway Analysis

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PBMCs were isolated by density gradient centrifugation and resuspended in R10. PBMCs were rested in RPMI without glutamine and FBS (R0) for two hours. In some cases, PBMC are incubated in R10 for 1–2 days, passed through a 40µµ strainer and adjusted to 0.5 × 10⁶/mL in PBS and rested prior to PMA stimulation. For glutamine supplementation experiments, PBMCs were rested in PBS containing 0.2mM glutamine and increasing concentrations of glutamine (0–5 mM) were added before PMA stimulation. PMA (1–20ng/mL) stimulation was performed in 1 mL PBS for 5–30 minutes in 37°C CO₂ incubator. After stimulation, cells were fixed in 1.6% paraformaldehyde for 10 minutes at room temperature, spun down and resuspended in 100% cold methanol and incubated overnight at −20°C. Cells were then washed and stained with fluorochrome-conjugated phospho-antibodies: phospho-S6 (N7–548, BD Biosciences), phospho-ERK1/2 (20A, BD Biosciences), phospho-P65 (K10–895.12.50, BD Biosciences), phospho-P38 (36/p38, BD Biosciences), phospho-AKT (pS473, M89-61, BD Biosciences), IκBα (L35A5, Cell Signaling Technology), along with CD3 (HIT3a, BioLegend), CD4 (L200, BD Biosciences), CD45RO (UCHL1, BD Biosciences), CD45RA (HI100, BD Biosciences) antibodies.

Ma C.A., Stinson J.R., Zhang Y., Abbott J.K., Weinreich M.A., Hauk P.J., Reynolds P.R., Lyons J.J., Nelson C.G., Ruffo E., Dorjbal B., Glauzy S., Yamakawa N., Arjunaraja S., Voss K., Stoddard J., Niemela J., Zhang Y., Rosenzweig S.D., McElwee J.J., DiMaggio T., Matthews H.F., Jones N., Stone K.D., Palma A., Oleastro M., Prieto E., Bernasconi A.R., Dubra G., Danielian S., Zaiat J., Marti M.A., Kim B., Cooper M.A., Romberg N.D., Meffre E., Gelfand E.W., Snow A.L, & Milner J.D. (2017). Germline hypomorphic CARD11 mutations in severe atopic disease. Nature genetics, 49(8), 1192-1201.

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Allogeneic T-cell Proliferation Assay with CB-DC

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After isolation of CD34+ cells, used for the generation of CB-DC, the resultant CD34-fraction was enriched for T-cells using anti-CD3 magnetic microbeads (Miltenyi Biotec). For MLR, allogeneic T-cells (1 × 10⁶/mL) were labelled with cell trace violet (5 µM; Invitrogen, Carlsbad, CA, USA) and co-cultured with CB-DC (2 × 10⁵/mL) in a 96-well round-bottom plate (Corning) at a stimulator:responder ratio of 1:5. Unstimulated cell trace violet-labelled cells served as a negative control. After 4 days, cells were stained with CD3 (UCHT1), CD8 (RPA-T8), CD197 (G043H7), and CD45RO (UCHL-1) from BD Bioscience and analyzed using a FACS Canto II (BD Bioscience). The T-cell proliferation analysis was performed using a proliferation tool in the Flowjo software (Becton, Dickinson and Company) providing the division index.

Plantinga M., van den Beemt D.A., Dünnebach E, & Nierkens S. (2021). CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine. Cancers, 13(15), 3818.

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Cd45ro uchl1

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7 protocols using cd45ro uchl1

Multiparametric Flow Cytometry for T Cell Phenotyping

Comprehensive Immunophenotyping of PBMCs

PBMC Signaling Pathway Analysis

Comprehensive Immunophenotyping of PBMCs

T-cell Cytotoxicity and Proliferation Assays

PBMC Signaling Pathway Analysis

Allogeneic T-cell Proliferation Assay with CB-DC

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