Cd5 fitc

Manufactured by BioLegend

Sourced in United States

CD5-FITC is a fluorochrome-conjugated antibody that binds to the CD5 antigen. CD5 is a cell surface glycoprotein expressed on a subset of T cells and B cells. The FITC (fluorescein isothiocyanate) fluorochrome is used to label the CD5 antibody, enabling detection and analysis of CD5-positive cells by flow cytometry.

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8 protocols using cd5 fitc

Characterization of IL-10+ B cells in CLL

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To characterize IL-10+ B cell, we thawed cryopreserved PBMCs from CLL patients and healthy controls and stimulated them with CpG (4 µg/ml) (positive control) or CXCL12 (250 ng/ml) (R&D) for 14–16 h. Phorbol myristate acetate (PMA; 50 ng/mL), ionomycin (250 ng/mL, Sigma Aldrich) and brefeldin A (10 µg/mL; Sigma-Aldrich) were added for the last 6 h of culture. Cells were harvested, washed, and incubated for 20 min at room temperature with a cocktail of anti-CD19-PE-cy7, CD5-FITC (BioLegend), and Live/Dead-Aqua (Invitrogen). Cells were then washed and fixed/permeabilized for 1 h at 4°C with foxp3 staining buffer (eBioscience) according to the manufacturer’s instructions. Cells were incubated for 30 min at room temperature with rat APC-conjugated anti-IL-10 or rat IgG2aК isotype antibodies (BD Biosciences). For blocking experiments, we performed the assay in the presence or absence of anti-CXCR4 blocking antibody (BioLegend), the STAT3 inhibitor cucurbitacin (0.05 µM) or lenalidomide (10 µM). To determine apoptosis, cells were stained with annexin V-FITC and 7-AAD using the Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. All data were acquired with BD LSRFortessa (BD Biosciences) and analyzed with FlowJo software version 10.

Shaim H., Estrov Z., Harris D., Hernandez Sanabria M., Liu Z., Ruvolo P., Thompson P.A., Ferrajoli A., Daher M., Burger J., Muftuoglu M., Imahashi N., Li L., Liu E., Alsuliman A.S., Basar R., Nassif Kerbauy L., Sobieski C., Gokdemir E., Kondo K., Wierda W., Keating M., Shpall E.J, & Rezvani K. (2018). The CXCR4–STAT3–IL-10 Pathway Controls the Immunoregulatory Function of Chronic Lymphocytic Leukemia and Is Modulated by Lenalidomide. Frontiers in Immunology, 8, 1773.

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Multiparametric Flow Cytometry Analysis of B Cell Subsets

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Cells were stained with panels of antibodies including anti-mouse CD19-pacific Blue, CD138-APC, CD21-PEcy7, CD5-FITC, CD43-PE, IgD-APCcy7, CD38-PE-Cy7, GL-7-Percp (BioLegend, San Diego, CA ), and IgM-APCcy7 (Miltenyi Biotec, Auburn, CA). For the human B cell phenotypes, the following antibodies were used: anti-CD19-pacific blue, anti-CD38-PECy7, anti- CD20-PE, anti-CD27-APCcy7 (BioLegend), and anti– human IgG-PE, anti– human IgA-APC (Miltenyi Biotec). BD Cytofix/ Cytoperm and BD Perm/Wash (BD) were used for IgA and IgG intracellular staining per manufacturers’ instructions. Data were analyzed on a BD Biosciences LSRII machine and Flowjo software 29.

Qu P., Wuest T., Min Y., Alevizos I., Young H.A, & Lin P.C. (2020). Natural Killer Cell Transcript 4 promotes the development of Sjögren’s Syndrome via activation of Rap1 on B cells. Journal of autoimmunity, 116, 102559.

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Isolation and Characterization of B Cells

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Heparinized blood samples were obtained from normal donors and patients enrolled in clinical research protocols approved by the Human Subjects Protection Committee of the Dana-Farber Cancer Institute (DFCI), at UCSD and the Mayo Clinic (CLL Research Consortium), and through the ICGC (42 (link)) after obtaining written informed consent. Treatment indication for all 21 patients in the discovery cohort was determined based on iwCLL criteria (12 (link)), (44 (link)). Peripheral blood mononuclear cells (PBMC) from normal donors and patients were isolated by Ficoll/Hypaque density gradient centrifugation. Mononuclear cells were used fresh or cryopreserved with 10% DMSO FBS and stored in vapor-phase liquid nitrogen until the time of analysis. CD19+ B cells from normal volunteers and CLL samples with WBC ≤50 × 10⁹/L were isolated by immunomagnetic selection (Miltenyi Biotec, Auburn CA) and stained with anti CD19 PE (BioLegend) prior to FACS sorting for live single cells in the presence of DAPI. MBL cells and naïve and memory B cells from age-matched healthy donors were isolated as follows: cryopreserved PBMCs were thawed and stained with anti CD19 PE, CD5 FITC, and CD27 APC (BioLegend). Cells were gated for naïve B cells (CD19+; CD27-; CD5), memory B cells (CD19+; CD27+; CD5-) or MBL (CD19+; CD5+) (Supplementary Figure 7a).

Kretzmer H., Biran A., Purroy N., Lemvigh C.K., Clement K., Gruber M., Gu H., Rassenti L., Mohammad A.W., Lesnick C., Slager S.L., Braggio E., Shanafelt T.D., Kay N.E., Fernandes S.M., Brown J.R., Wang L., Li S., Livak K.J., Neuberg D.S., Klages S., Timmermann B., Kipps T.J., Campo E., Gnirke A., Wu C.J, & Meissner A. (2020). Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy. Blood Cancer Discovery, 2(1), 54-69.

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Analyzing STAT3 Phosphorylation in CLL and T Cells

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To analyze S727-STAT3 phosphorylation in CLL cells, we cultured PBMCs from CLL patients in the presence or absence of the STAT3 inhibitor cucurbitacin (0.05 µM) for 2 h or lenalidomide (10 µM) for 2 or 24 h at 37^oC. Cells were stained for 30 min with Live/Dead-Aqua (Invitrogen). Samples were stimulated with 250 ng/ml of CXCL12 for 20 min at 37^oC, and then fixed for 10 min in the dark. After one wash and 20 min of surface staining with CD19-APC (BD Biosciences) and CD5-FITC (BioLegend) antibodies, the cells were washed, permeabilized (Phospho-Epitopes Exposure kit-Beckman Coulter kit), and stained with p-S727-STAT3-PE mAb Phosflow antibody (BD Biosciences) for 30 min at room temperature.
To analyze Y705-STAT3 phosphorylation in T cells, we incubated healthy donor PBMC for 20 min at 37^°C with IL-10 (10 ng/ml) (R&D) or with supernatants collected from CXCL12-treated CLL cells (supernatant was harvested after CLL cells were treated with 250 ng/ml of CXCL12 for 48 h). Cells were fixed and stained with anti-CD3-v450 (BD Biosciences) and phosphorylated Y705-STAT3-PE mAb Phosflow antibody (BD Biosciences), following the protocol described above. To examine the effect of lenalidomide on Y705-STAT3 phosphorylation in T cells we pre-incubated the T cells for 2 h with lenalidomide (10 µM) before adding IL-10/CLL cells supernatant.

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CLL-RS Cell Sorting Protocol

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For suspension samples with admixture of both CLL and RS cells, cells were thawed by drop-wise addition of warmed media (RPMI 10% FCS) and stained with antibodies (Biolegend CD5 FITC cat#364022, CD19 PE-Cy7 cat#302216, CD3 PB cat#300330 using 2–4 uL of each antibody per 100 uL test) and a viability marker (Biolegend 7-AAD cat#420404 at 1:500 or Zombie Violet cat#423114 at 1:1000) before resuspension in PBS-0.04% BSA (Ultrapure NEB/Invitrogen). For Patients 19 and 41, viable CD5+ CD19+ cells were sorted into RS and CLL fractions by size based on the increased forward scatter (FSC) of RS cells (BD FACS Aria II). For Patients 43, 4 and 10, viable cells within the lymphocyte gate were sorted for analysis.

Stefanovsky V.Y., Pelletier G., Bazett-Jones D.P., Crane-Robinson C, & Moss T. (2001). DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules. Nucleic Acids Research, 29(15), 3241-3247.

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STAT3 Phosphorylation Profiling

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Cells were stained with Live/Dead Aqua (Life Technology), CD19-V450 (BD) and CD5-FITC (BioLegend) Abs for 20 minutes, washed, fixed/permeabilized (PerFix EXPOSE, Beckman Coulter) and stained with the p-S727-STAT3-PE mAb (BD Biosciences) for 30 minutes at room temperature.

Kondo K., Shaim H., Thompson P.A., Burger J.A., Keating M., Estrov Z., Harris D., Kim E., Ferrajoli A., Daher M., Basar R., Muftuoglu M., Imahashi N., Alsuliman A., Wierda W., Jain N., Liu E., Shpall E.J, & Rezvani K. (2017). Ibrutinib modulates the immunosuppressive CLL microenvironment through STAT3-mediated suppression of regulatory B cell function and inhibition of the PD-1/PD-L1 pathway. Leukemia, 32(4), 960-970.

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CLL Patients' PBMC Stimulation and Analysis

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Cryopreserved PBMCs from CLL patients collected before, and 3 and 6 months after ibrutinib therapy were thawed and stimulated with CpG (4μg/ml; Hycult biotech) plus CD40L (100 ng/mL) for 10 hours. In another experiment, PBMCs from untreated CLL patients were cultured with ibrutinib (1μM) or the STAT3 inhibitor cucurbitacin overnight (0.05 μM) and stimulated with CpG and CD40L for 10 hours. Phorbol-myristate acetate (PMA) (50 ng/mL; Sigma-Aldrich), ionomycin (250 ng/mL; Sigma-Aldrich), and brefeldin-A (10 μg/mL) were added for the last 6 hours of the culture. Cells were then washed and incubated for 20 minutes with anti-CD19-PE-cy7, CD5-FITC (BioLegend) and Live/Dead Aqua (Life technology). Cells were then washed and fixed/permeabilized for 1 hour at 4°C using a Foxp3 staining buffer set (eBioscience). Cells were incubated for 30 minutes at room temperature with APC-conjugated IL-10 or IgG2aK isotype antibodies (BD). All data were acquired with BD-Fortessa (BD Biosciences) and analyzed with FlowJo software.

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Phospho-Erk1/2 Analysis in Thymocytes

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2 × 10⁶ thymocytes were left for 10 min at 37 °C and stained with CD4-PE-Cy5, CD8-APC-Cy7, CD5-FITC, CD69-PE, and TCRβ-Pacific Blue (BioLegend, San Diego, CA, USA). Subsequently, cells were fixed and permeabilized for 10 min with 3.7% PFA (ChemCruz Biochemicals, Santa Cruz, CA, USA) + 0.2% saponin (Sigma Aldrich, Saint Louis, MO, USA). Subsequently, samples were washed and incubated with a phospho-specific Erk1/2 antibody (Cell Signaling, Danvers, MA, USA) in PBS containing 0.1% saponin, 0.1% sodium azide, and 0.2% BSA. Cells were washed and stained with APC-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Samples were analyzed on a FACS Fortessa I (BD Biosciences, Franklin Lakes, NJ, USA).

Kästle M., Merten C., Hartig R., Plaza-Sirvent C., Schmitz I., Bommhardt U., Schraven B, & Simeoni L. (2022). Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection. International Journal of Molecular Sciences, 23(13), 7271.

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