Cd127 fitc

Manufactured by BD

Sourced in United States, Poland, United Kingdom

CD127-FITC is a fluorescently labeled monoclonal antibody that binds to the human CD127 (IL-7 receptor alpha chain) surface marker. It is used for the identification and quantification of CD127-expressing cells in flow cytometry applications.

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Lab products found in correlation

Cd25 apc, by BD (4 mentions) Cd4 v500, by BD (3 mentions) Cd25 pe, by BD (2 mentions) Foxp3 pe molecules, by BD (2 mentions) Cd4 percp, by BD (2 mentions) Cd45 apc h7, by BD (2 mentions) Cd8 apc h7, by BD (2 mentions) Fixable live dead marker, by Thermo Fisher Scientific (2 mentions) Moflo xdp high speed cell sorter, by Beckman Coulter (2 mentions) Cd8 pe cf594, by BD (2 mentions) Cd3 alexa fluor 750, by Bio-Rad (2 mentions) Cd4 fitc, by BD (1 mentions) Cd8 pe, by BD (1 mentions) Imk plus kit, by BD (1 mentions) Facs lysing solution, by BD (1 mentions) Facscalibur, by BD (1 mentions) Cd4 pacific blue clone rpa t4, by BD (1 mentions) Live dead staining, by Thermo Fisher Scientific (1 mentions) Cell wash buffer, by BD (1 mentions) Cd34 pe, by BD (1 mentions)

14 protocols using cd127 fitc

T-cell Subtype Characterization by Flow Cytometry

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Key T-cell subtypes surface markers and activity markers relative expression levels evaluated by flow cytometry method.
At 4^th, 6^th, 10^th days the cells harvested from each concentration group, 200-500 × 10³ cells prepared for CD markers analyzing. CD4-FITC (as Helper T marker), CD8-PE (as Cytotoxic T marker), CD25-PE, FOXP3-PerCp (as regulatory T-cell marker together with CD4) and CD127-FITC (as memory differentiation of T-cells) (BD, Bioscience, USA) based on single or triple panel orders. Briefly 3-5 μL of respective antibody-incubated for 45 minutes to one hour with cells in darkness and washed by PBS, the pellet resuspended in 500-1000 μL PBS and analyzed by BD-FACSCalibur flowcytometry systems. For intracellular markers, permeabilization steps before antibody steps have been done based on manufacturer instructions.

Talebi M., Nozad Charoudeh H., Movassaghpour Akbari A.A., Baradaran B, & Kazemi T. (2020). Effect of Cellular-Based Artificial Antigen Presenting Cells Expressing ICOSL, in T-cell Subtypes Differentiation and Activation. Advanced Pharmaceutical Bulletin, 11(3), 537-542.

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Lymphocyte Immunophenotyping and Treg Analysis

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Analysis of lymphocytes phenotypes was performed as previously described [11 ]. Peripheral blood samples were collected in EDTA-anticoagulated tube. Determination of blood lymphocyte immunophenotype was evalutaed by IMK Plus Kit (BD Biosciences, Poland) after erythocyte lysis (BD FACS Lysing Solution, BD Biosciences, Poland). Stained cells were acquired in flow cytometry (Facs Calibur, BD, Germany). Additionally percentage distributions of white blood cells (lymphocytes, monocytes, granulocytes, neutrophils, eosinophils) was done using CD45, FITC, CD14 PE antibody and FSC/SSC determination. Results of lymphocyte phenotypes are presented as mean percentage of lymphocytes ± SD.
To characterize natural T regulatory cells, cells were stained with primary antibodies against CD4-PerCP, CD25-APC, CD127-FITC and FoxP3 PE molecules (BD Bioscence, Poland) or appropriate isotype control. Cells were acquired in flow cytometry. 10 000 counts of CD4 PerCP positive cells finished the acquisition. Percentage of nTreg cells (FoxP3⁺, CD25^high, CD127^low/–) were counted in CD4 postive cells.

Wawrzyniak A., Lipińska-Opałka A., Zdanowski R., Lewicki S., Murawski P, & Kalicki B. (2017). Evaluation of selected immunological parameters and the concentration of vitamin D in children with asthma. Case-control study. Central-European Journal of Immunology, 42(1), 101-106.

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Identifying T Helper Cell Subsets

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We also considered the frequency of different T helper (Th1, Th2, Th17, and Treg) subset populations using flow cytometry. We resuspended the cells in PBS in flow-cytometry staining tubes. We used live/dead staining (Molecular Probes/Invitrogen) in all our samples and identified live cells by the intracellular conversion of a calcein ester to free calcein, which is fluorescent in the green spectrum; we identified dead cells by the red staining of internal nucleic acids using ethidium homodimer. We further stained the fraction with CD4-Pacific Blue (clone RPA-T4, BD Biosciences), CD25-phycoerythrin (PE)-Cy7 (clone BC96, BioLegend), and CD127-FITC (BD Biosciences). We gated CD127 low-expressing CD4 + CD25hi cells to ensure separation of conventional activated T cells from stable Treg populations, which further allowed us to identify highly-enriched FoxP3 + cells [25 (link)].

Aguilera J., Han X., Cao S., Balmes J., Lurmann F., Tyner T., Lutzker L., Noth E., Hammond S.K., Sampath V., Burt T., Utz P.J., Khatri P., Aghaeepour N., Maecker H., Prunicki M, & Nadeau K. (2022). Increases in ambient air pollutants during pregnancy are linked to increases in methylation of IL4, IL10, and IFNγ. Clinical Epigenetics, 14, 40.

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Multiparameter Flow Cytometry of Bone Marrow

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Flow cytometry analysis was performed immediately after bone marrow collection. Cells were incubated with appropriate antibodies for 20 min at room temperature. To remove erythrocytes, the cells were incubated with BD Pharm Lyse lysing buffer (BD Biosciences). After being washed in Cell Wash Buffer (BD Biosciences), the cells were suspended in Cell Wash Buffer and analyzed using a BD FACSCanto flow cytometer (BD Biosciences). Gate parameters dividing negative from positive cells were chosen based on isotype antibody control probes.
Tregs were detected based on the CD4 þ CD25 high CD127 - phenotype using the following antibodies: CD4-PerCP, CD25-APC, CD127-FITC (BD Biosciences). We always calculated the percentage of Tregs among all CD4 þ cells. Subpopulations of HSCs (CD45 þ CD34 þ phenotype) exhibiting high expression of CD274 or CD47 markers were identified using CD34-PE, CD45-PerCP, CD47-Alexa Fluor 647, and CD274-FITC antibodies (all antibodies were obtained from BD Biosciences).

Ciomber A., Mitrus I., Fidyk W., Smagur A., Chwieduk A., Glowala-Kosinska M., Czerw T., Sobczyk-Kruszelnicka M., Mendrek W., Sadus-Wojciechowska M., Najda J., Holowiecki J, & Giebel S. (2016). Immunological properties of bone marrow microenvironment 1 year after allogeneic hematopoietic stem cell transplantation. Experimental hematology, 44(12).

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Isolation and Evaluation of Regulatory T Cells

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Heparinized peripheral blood obtained from patients was layered onto a Ficoll-Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA) density gradient and centrifuged at 2,000 rpm for 30 minutes. Mononuclear cells were collected at the interfaces, washed and re-suspended in Phosphate Buffer Saline (PBS), supplemented with 2mM EDTA, and further enriched with CD4+ lymphocytes by negative selection using an appropriate CD4+ T cells isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany).
For Tregs evaluation, CD4+ lymphocytes were stained with an Antibody cocktail containing CD4+ PerCP Cy5.5, CD25 PE and CD127 FITC (all from BD Biosciences Pharmingen, San Diego, CA, USA), for 20 minutes at room temperature, and subsequently fixed and permeabilized for intranuclear FoxP3 expression with an anti-human Foxp3-APC staining set (eBioscience, San Diego, CA, USA), accordingly to manifacturer’s instructions. Acquisitions were made on BD-FACSCanto II Flow Cytometer, equipped with FACS Diva Software; Treg cells were identified in the CD4+ CD25 high, CD127^_ Foxp3+ cells fraction and expressed as percentage of total CD4+ lymphocytes.

Bonanni A., Bertelli R., Rossi R., Bruschi M., Di Donato A., Ravani P, & Ghiggeri G.M. (2015). A Pilot Study of IL2 in Drug-Resistant Idiopathic Nephrotic Syndrome. PLoS ONE, 10(9), e0138343.

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Comprehensive Peripheral Blood Lymphocyte Analysis

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The analysis was carried out as described before [6] . To assess the phenotype of the peripheral blood lymphocytes, the antibodies from the IMK plus test were added to the appropriate cytometric tubes (IMK Test, BD Biosciences, Poland): A -CD45 / CD14, B -isotype control, C -CD3 / CD19, D -CD4 / CD8, E -CD3 / anti-HLA-DR, F -CD16 / 56 / CD3. After adding the erythrocytes lysis solution, the samples were rinsed with buffered saline and subjected to the cytometric analysis.
To characterize natural T-regulatory lymphocytes, cells were stained with antibodies against CD4-PerCP, CD25-APC, CD127-FITC and FoxP3 PE molecules (BD Bioscience, Poland). Cells were collected by ow cytometry. The percentage of nTreg cells (FoxP3 +, CD25high, CD127) was estimed in the CD4 + cell population.
To analyze the cytokines, the 50 µl beads, 50 µl tested serum and 50 µl detection reagent were added to the cytometric tubes. The samples were incubated for 3 hours at room temperature, in dark. Subsequently, each sample was rinsed and centrifuged, and the supernatant was collected and washed with 300 µl of washing buffer. The cytokine level was analyzed using a FACS Calibur ow cytometer.
The measured cytokines were divided into the pro-in ammatory or anti-in ammatory category:
• pro-in ammatory: IL-1, IL-2, IL-6, IL-17A, IL-22, TNFα, IFNγ,
• anti-in ammatory: IL-4, IL-10, TGF-β.

Wawrzyniak A., Lipińska-Opałka A., Kalicki B, & Kloc M. (2021). The Effect of Passive Exposure to Tobacco Smoke on the Immune Response in Children with Asthma. Substance use & misuse, 56(3).

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Flow Cytometry Analysis of Regulatory T Cells

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For extracellular staining, harvested cells were washed and incubated in PBS containing 1% FBS containing the below fluorochrome-conjugated antibodies in a flow tube. For intracellular staining of cytokines, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml, Biogems), ionomycin calcium salt (1 μg/ml, Biogems), and brefeldin A (5 μg/ml, Biogems) for 6 h. Then, cells were stained with surface markers and further fixed/permeabilized (BioLegend) and stained for intracellular protein. Human-specific monoclonal antibodies used for flow cytometry included CD4 (PE/cy7), CD25 (APC/cy7), Foxp3 (AF647), IL-10 (PE), TGF-β1 (AF488), CD45RA (APC), Annexin V (Pacific blue), propidium iodide (PI) purchased from BioLegend, and CD127 (FITC) purchased from BD Pharmingen. Sample detection was performed by MACSQuant Analyzer 10 (Miltenyi Biotec, Germany) and data were analyzed with FlowJo software.

Gan X., Zhang R., Gu J., Ju Z., Wu X., Wang Q., Peng H., Qiu J., Zhou J., Cheng F, & Lu L. (2020). Acidic Microenvironment Regulates the Severity of Hepatic Ischemia/Reperfusion Injury by Modulating the Generation and Function of Tregs via the PI3K-mTOR Pathway. Frontiers in Immunology, 10, 2945.

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Multiparametric Immune Cell Profiling

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For the study of the cell populations, the following monoclonal antibodies were used: Granulocyte/macrophage-colony stimulating factor (GM-CSF)-PE, CD197-PE (CCR7-PE), CD24-PE, Interferon (IFN)-gamma-FITC, CD14-FITC, CD16-PE-Cy7, CD8-FITC, CD27-FITC, CD8-APC-H7, CD4-APC-H7, CD4-PE-Cy7, CD123-Bv421, CD56-APC, CD56-Bv421, CD45RO-APC, CD45-APC-H7, CD3-BV421, CD127-BV421, CD127-FITC, CD45-V500 TNF-alpha-PerCP-Cy5.5, CD38-PE-Cy5.5, CD19-PE-Cy7, CD19-APC, CD25-PE-Cy7, CD3-PerCP, HLA-DR-PerCP, CD11c-PE (BD Biosciences, San Diego, CA, USA) and IL-17-APC (R&D Systems, Minneapolis, MN, USA).

Dominguez-Mozo M.I., Perez-Perez S., Villarrubia N., Costa-Frossard L., Fernandez-Velasco J.I., Ortega-Madueño I., Garcia-Martinez M.A., Garcia-Calvo E., Estevez H., Luque Garcia J.L., Torrejon M.J., Arroyo R., Villar L.M, & Alvarez-Lafuente R. (2021). Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls. Cells, 10(1), 119.

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Comprehensive Immune Cell Profiling

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The following conjugated antibodies were used for cell-surface staining: CD3-Pacific Blue (BD Pharmingen; Clone UCHT1; Cat. No. 558117; San Diego, CA, USA), CD8-APC H7 (BD Pharmingen; Clone SK1; Cat. No. 641400), CD45RA-PE (BD Pharmingen; Clone HI100; Cat. No. 555489), CCR7-PE-Cy7 (BD Pharmingen; Clone 3D12; Cat. No. 557648), CD28-PerCP-Cy5.5 (BD Biosciences; Clone L293; Cat. No. 337181; San Jose, CA, USA), CD27-Alexa Fluor 700 (BD Pharmingen; Clone M-T271; Cat. No. 560611), CD95-APC (BD Pharmingen; Clone DX2; Cat. No. 558814) and CD127-FITC (BD Pharmingen; Clone HIL-7R-M21; Cat. No. 560549). Conjugated antibodies for intracellular staining included the following: IFN-γ-FITC (BD Pharmingen; Clone 4S.B3; Cat. No. 554551), IL-2-PerCP-Cy5.5 (BD Pharmingen; Clone MQ1-17H12; Cat. No. 560708) and TNF-α-AlexaFluor 700 (BD Pharmingen; Clone MAb11; Cat. No. 557996). To exclude dead cells, the Fixable Aqua Dead Cell Stain viability marker was used (Invitrogen; Cat. No. L34957; Eugene, OR, USA).

Mateus J., Lasso P., Pavia P., Rosas F., Roa N., Valencia-Hernández C.A., González J.M., Puerta C.J, & Cuéllar A. (2015). Low Frequency of Circulating CD8+ T Stem Cell Memory Cells in Chronic Chagasic Patients with Severe Forms of the Disease. PLoS Neglected Tropical Diseases, 9(1), e3432.

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Flow Cytometric Analysis of nTreg Cells

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Analysis of nTreg cells was performed as previously described^{52 (link)}. Briefly, blood samples (100 µl) were stained with primary antibodies CD4-PerCP, CD25-APC, CD127-FITC (extracellular staining, BD Bioscence, Poland), or appropriate isotype control with additionally CD4-PerCP antibody. Blood samples were incubated 20 min at room temperature in the dark, and then erythrocytes lysis was performed. After double PBS wash, cells were fixed and permeabilized in Fixation/Permeabilization buffer (BD Pharmingen, Poland) and stained with FoxP3 PE or isotype IgG1 kappa PE antibody (45 min at room temperature in the dark). Afterward, cells were washed twice with PBS, fixed in 300 μl of 1% PFA in PBS solution, and analyzed by flow cytometry. The acquisition was stopped after 10,000 counts of CD4 PerCP positive cells. We defined natural Treg (nTreg) cells as: CD4⁺/CD25^high/CD127^low/FoxP3⁺.

Szymański Ł., Urbańska W., Ciepielak M., Cios A., Stankiewicz W., Stelmasiak M., Rzeszotarska A., Korsak J., Lewicki S, & Chciałowski A. (2022). Time-dependent effect of desensitization with wasp venom on selected parameters of the immune system. Scientific Reports, 12, 7206.

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