Cd127 fitc, by Thermo Fisher Scientific - Product details

1

Profiling T-cell activation and pSTAT5 signaling

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Flow cytometry and basal pStat5 experiments were performed according to previously published methods [17 (link), 41 (link)]. Monoclonal antibodies (mAbs) used were CD3-APCa750, CD4-ECD, CD8-APCa700, CD16-FITC, CD19-ECD, CD45RO-FITC, CD56-PCy7 and CD152-PE from Beckman Coulter; CD25-PE, CD45RA-APC and CD45RA-PCy7 from BD Biosciences; CD127-FITC from eBioscience; CD25-APC and LAP-PE from R&D Systems and GITR-PE from Miltenyi.
For pSTAT5 activation experiments, peripheral blood mononuclear cells (PBMCs) were cultured (500,000/well) for 30 min at 37° C and IL-2 was added for 15 min. Cells were then fixed with 1.6% paraformaldehyde, permeabilized with 100% methanol, stained for pSTAT5 and appropriate surface markers. Events were collected using a LSRFortessa flow cytometer (BD Biosciences) and data were analyzed using Diva software (BD Biosciences). To calculate EC50, after subtracting the value from the media control, binding data for each sample was normalized to 100% based on the maximal response and non-linear regression of these data was performed assuming a variable slope using Graph-Pad Prism 6.0.

Rosenzwajg M., Churlaud G., Mallone R., Six A., Derian N., Chaara W., Lorenzon R., Long A., Buckner J., Afonso G., Pham H.P., Hartemann A., Yu A., Pugliese A., Malek T.R, & Klatzmann D. (2015). Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. Journal of autoimmunity, 58, 48-58.

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2

Multiparameter Analysis of Tonsillar Lymphocytes

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Tonsillar lymphocytes were stained with the following anti-human
antibodies – CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or
Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421
(EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5,
BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD
Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences),
CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC
or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD
Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience),
CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605
(SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1,
BD Bioscience). All surface stains were performed in the presence of Human
TruStain FcX (cat. 422302, BD Bioscience). Intracellular staining was performed
using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) according
to the manufacturer’s instructions. Cells were stained with primary
antibodies followed by secondary reagents for 30 min at 4 °C. Data were
collected on a LSRII or Fortessa cytometer (BD) and analysed with FlowJo
software (TreeStar). 7-AAD (Invitrogen) or Zombie Aqua (BioLegend) staining was
used to exclude dead cells from analysis.

Papa I., Saliba D., Ponzoni M., Bustamante S., Canete P.F., Gonzalez-Figueroa P., McNamara H.A., Valvo S., Grimbaldeston M., Sweet R.A., Vohra H., Cockburn I.A., Meyer-Hermann M., Dustin M.L., Doglioni C, & Vinuesa C.G. (2017). TFH-derived dopamine accelerates productive synapses in germinal centres. Nature, 547(7663), 318-323.

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3

Multicolor Flow Cytometry for T and B Cell Subsets

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PBMC were isolated from peripheral blood by Ficoll gradient and frozen for batched analysis. We designed six multicolor flow cytometry panels to quantify 60 T cell subsets along with two B cell subsets, and calculated the CD4⁺/CD8⁺ T cell ratio (Supplementary Table 1). The following fluorochrome-conjugated anti-human antibodies were used from BD Biosciences (San Jose, CA): CD3-FITC, CD3-PerCP-Cy5.5, CD4-APC, CD4-APC-Cy7, CD8-BV510, CD45RO-FITC, CD45RA-APC, CD45RA-APC-Cy7, CCR4-PE, CD27-PE, CD28-BV421, CD138-BV421, CCR6-BV421, CXCR3-PE, CCR7-A700, IL-17-BV786, IFN-γ-PE-Cy7, iso IgG1k-FITC, iso IgG1k-PE-Cy7, iso IgG2bk-APC, iso IgG1k-APC-Cy7, and iso IgG1k-BV510; from Biolegend (San Diego, CA): CD127-FITC, CD27-APC, CD57-PerCp-Cy5.5, CD19-BV510, PD-1-APC-Cy7, CXCR5-FITC, and TNFα-FITC; from eBioscience (San Diego, CA): CD4-PE-Cy7, and IL-2-PE; from Miltenyi Biotec (San Diego, CA): CD25-APC and KLRG1-PE; from Beckman Coulter (Brea, CA): CD38-PE-Cy7.

Hartzell S., Bin S., Cantarelli C., Haverly M., Manrique J., Angeletti A., Manna G.L., Murphy B., Zhang W., Levitsky J., Gallon L., Yu S.M, & Cravedi P. (2020). Kidney Failure Associates With T Cell Exhaustion and Imbalanced Follicular Helper T Cells. Frontiers in Immunology, 11, 583702.

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4

Multi-parameter Flow Cytometry of Lymphocyte Subsets

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Multi-parameter flow cytometry analysis of different cell subsets was performed using PBMC from patients at baseline and at cycle 3. Cells were stained with different antibody panels to study different lymphocyte populations. To study T regulatory (Treg) cells we used the following panel: CD3-AmCyan, Foxp3-efluor450 (eBioscience, CA), CD127-FITC, ICOS-PE (eBioscience, CA), CD4-PerCP-Cy5.5, CD39-APC, CD25-PE-Cy7 and CD8-APC-H7. For the proliferation panel we stained for CD3-AmCyan, Foxp3-efluor450, KI-67-Alexa Fluor 488, ICOS-PE, CD4-PerCP-Cy5.5, CD39-APC, CD25-PE-Cy7 and CD8-APC-H7. The frequencies of the different populations were translated into cell numbers (# cells/µl of blood) using Absolute Lymphocyte Counts (ALC). All antibodies are from BD Biosciences, unless otherwise indicated. The acquisition was carried out on a FACS Canto II flow cytometer (BD Biosciences, CA). All analysis was done with the software FlowJo (Tree Star, OR).

Plimack E.R., Desai J.R., Issa J.P., Jelinek J., Sharma P., Vence L.M., Bassett R.L., Ilagan J.L., Papadopoulos N.E, & Hwu W.J. (2014). A phase I study of decitabine with pegylated interferon α-2b in advanced melanoma: impact on DNA methylation and lymphocyte populations. Investigational new drugs, 32(5), 969-975.

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5

Multiparametric Flow Cytometry Immunophenotyping

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Adherent cells or EBs were dissociated to form a single-cell suspension by TrypLE (Life Technologies) treatment, and washed with FACS buffer (1% FBS and 1 mM EDTA in PBS). The dissociated cells were resuspended in FACS buffer, and labeled with fluorochrome-conjugated anti-human CD34-APC (clone# AC136, Miltenyi Biotec), CD31-PE (clone# AC114.5, Miltenyi Biotec), CD144-Alexa Fluor 700 (clone# 16B1, eBioscience), CD45-BV605 (clone# HI30, BioLegend), CD41-APC/Cy7 (clone# HIP8, BioLegened), CD235a-PE (clone# HIR2, eBioscience), CD43-BV421 (clone# 1G10, BD Biosciences), CD73-PE/Cy7 (clone# AD2, BioLegend), CD117-PE/Cy7 (clone# 104D2, BioLegend), CD127-FITC (clone# eBioRDR5, eBioscience), CD14-FITC (clone# 61D3, eBioscience), or KDR-PerCP (clone# HKDR-1, BioLegend). Flow cytometry was performed on LSR II analyzer (BD Biosciences). Data analysis was performed using FlowJo software or FCS Express software.

BAI H., LIU Y., XIE Y., HOYLE D.L., BRODSKY R.A., CHENG L., CHENG T, & WANG Z.Z. (2015). Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells. Journal of cellular physiology, 231(5), 1065-1076.

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6

Phenotyping OVA-specific CD8+ T cells

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Splenocytes prepared as described previously were stained with H-2K^b-OVA₂₅₇ MHC I pentamers (ProImmune, Oxford, UK) in room temperature FACS buffer for 10 minutes in the presence of 2-4G2 antibody. Cells were washed once and stained with surface antibodies plus L/D NIR for 20 minutes on ice. Antibodies included CD127-FITC, CD44-PerCP-Cy5.5, KLRG1-APC, CD8-Alexa Fluor 700 (all from eBioscience), and B220-V500 (BD). Cells were washed, fixed with Cytofix, and analyzed as previously. Within the viable CD8 T-cell population, CD44^hi H-2K^b-OVA₂₅₇ pentamer⁺ events, gated based on the 99.9th percentile in unimmunized mice, were analyzed for their expression of KLRG1 and CD127.

Odegard J.M., Kelley-Clarke B., Tareen S.U., Campbell D.J., Flynn P.A., Nicolai C.J., Slough M.M., Vin C.D., McGowan P.J., Nelson L.T., ter Meulen J., Dubensky TW J.r, & Robbins S.H. (2015). Virological and Preclinical Characterization of a Dendritic Cell Targeting, Integration-deficient Lentiviral Vector for Cancer Immunotherapy. Journal of Immunotherapy (Hagerstown, Md. : 1997), 38(2), 41-53.

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7

Flow Cytometry Analysis of T Cell Responses

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Standard flow cytometry was used as described in [16 (link)] for testing IFN-γ expression and CD8⁺/CD4⁺ T cell memory response. The following conjugated antibodies from eBiosciences (San Diego, CA, USA) were used: CD3-APC (145-2C11), CD4-PE (GK1.5), CD8-PerCP (53-6.7), CD44-AF780 (IM7), CD127-FITC, and CD44^hiCD127⁺. After washing, splenocytes were fixed before being measured on a BD LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software 10.0 (Tree Star, Ashland, OR, USA). The MHC class II tetramers, which present AS15 to T cells, were obtained from the NIH tetramer facility and used as described previously.

El Bissati K., Krishack P.A., Zhou Y., Weber C.R., Lykins J., Jankovic D., Edelblum K.L., Fraczek L., Grover H., Chentoufi A.A., Singh G., Reardon C., Dubey J.P., Reed S., Alexander J., Sidney J., Sette A., Shastri N, & McLeod R. (2023). CD4+ T Cell Responses to Toxoplasma gondii Are a Double-Edged Sword. Vaccines, 11(9), 1485.

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8

Multiparameter Flow Cytometry Immunophenotyping

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Fresh total cells from the respective tissues were directly stained with the following monoclonal antibodies (mAbs) at predetermined optimal dilutions for 20 min at 4°C: CD3-PE, CD8-Alexa700, CD4-HorizonV500, CD127-FITC, and CD25-PeCy7 (eBioscience). Intracellular detection of FOXP3 (FOXP3-E450, eBioscience), CTLA-4 (CTLA-4-APC, eBioscience), Ki-67 (Ki-67-FITC, BD Pharmingen), and Bcl-2 (Bcl-2-PE, BD Pharmingen) was performed on fixed and permeabilized cells using appropriate buffer (eBioscience) (incubation 30 min at 4°C). Cells were acquired on an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded by forward/side scatter gating.

Churlaud G., Pitoiset F., Jebbawi F., Lorenzon R., Bellier B., Rosenzwajg M, & Klatzmann D. (2015). Human and Mouse CD8+CD25+FOXP3+ Regulatory T Cells at Steady State and during Interleukin-2 Therapy. Frontiers in Immunology, 6, 171.

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Multicolor Flow Cytometry Gating Strategy

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Multicolor flow cytometry gating strategy was applied to analyze various cell populations simultaneously. Expanded cells were co-stained with 6 different antibodies and analyzed with LSRII flow cytometer (BD Bioscience, San Jose, CA). Briefly, cells were re-suspended in 100 μl MACS buffer (Miltenyi Biotec, Auburn, CA) and stained with antibody cocktail in the dark at 4°C for 15 minutes. Washing twice with MACS buffer was performed before and after cell staining. The antibody cocktail contains: FITC-conjugated anti-Vδ2 TCR (Biolegend, San Jose, CA), APC-conjugated anti-CD3 (Biolegend), PE conjugated anti-CD4 (BD), PE-CyC7-conjugated anti-CD56, APC-CyC7-conjugated anti-CD16, V450-conjugated anti-CD8a Beckman Coulter (Fullerton, CA). The antibody cocktail used for Treg analysis contains CD4-PE, CD25-APC (BD Bioscience, San Jose, CA), and CD127-FITC (eBioscience) to detect CD4+CD25+CD127- Treg population. The gating strategies used in the current study are included in S1 and S2 Figs. Cell populations were also analyzed with one- or two-color flow cytometry in Accuri C6 flow cytometer (BD). FITC-anti-Vδ2 TCR, APC-anti-CD3, and PE-anti-CD3, and APC-anti-CD56 (Biolegend) were used.

Du S.H., Li Z., Chen C., Tan W.K., Chi Z., Kwang T.W., Xu X.H, & Wang S. (2016). Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy. PLoS ONE, 11(9), e0161820.

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Multiparameter Analysis of Tonsillar Lymphocytes

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Tonsillar lymphocytes were stained with the following anti-human
antibodies – CD4 APCCy7 (RPA-T4, BD Biosciences), CXCR5 Alexa 488 or
Alexa 647 (RF8B2, BD Biosciences), PD-1 PE (MIH4, eBioscience) or BV605 or BV421
(EH12.2H7, BioLegend), CD127 FITC (11-1278, eBioscience) or BV 421 (A019D5,
BioLegend), CD25 biotin (BC96, eBioscience or BioLegend) or PE-Cy7 (BC96, BD
Biosciences or BioLegend), BCL6 Alexa 647 or PE-Cy7 (K112-91, BD Biosciences),
CD3 APC (HIT3a, BD Biosciences) or Alexa 700 (UCHT1, BD Biosciences), CD27 FITC
or APC (M-T271, BD Biosciences), CD38 FITC (HIT2, BD Biosciences) or PE (HB7, BD
Biosciences), ICOSL APC (2D3, BioLegend), FAS PE-CF594 (DX2, BD Bioscience),
CD40 APCCy7 (5C3, BioLegend), BAFFR PECy7 (11C1, BioLegend), CD19 PECy7 or BV605
(SJ25C1, BD Bioscience), IL21R BV421 (17A12, BioLegend), CD86 BV421 (2331/FUN-1,
BD Bioscience). All surface stains were performed in the presence of Human
TruStain FcX (cat. 422302, BD Bioscience). Intracellular staining was performed
using the FOXP3/Transcription Factor Staining Buffer Set (eBioscience) according
to the manufacturer’s instructions. Cells were stained with primary
antibodies followed by secondary reagents for 30 min at 4 °C. Data were
collected on a LSRII or Fortessa cytometer (BD) and analysed with FlowJo
software (TreeStar). 7-AAD (Invitrogen) or Zombie Aqua (BioLegend) staining was
used to exclude dead cells from analysis.

Papa I., Saliba D., Ponzoni M., Bustamante S., Canete P.F., Gonzalez-Figueroa P., McNamara H.A., Valvo S., Grimbaldeston M., Sweet R.A., Vohra H., Cockburn I.A., Meyer-Hermann M., Dustin M.L., Doglioni C, & Vinuesa C.G. (2017). TFH-derived dopamine accelerates productive synapses in germinal centres. Nature, 547(7663), 318-323.

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Cd127 fitc

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12 protocols using cd127 fitc

Profiling T-cell activation and pSTAT5 signaling

Multiparameter Analysis of Tonsillar Lymphocytes

Multicolor Flow Cytometry for T and B Cell Subsets

Multi-parameter Flow Cytometry of Lymphocyte Subsets

Multiparametric Flow Cytometry Immunophenotyping

Phenotyping OVA-specific CD8+ T cells

Flow Cytometry Analysis of T Cell Responses

Multiparameter Flow Cytometry Immunophenotyping

Multicolor Flow Cytometry Gating Strategy

Multiparameter Analysis of Tonsillar Lymphocytes

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