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Leukocyte activation cocktail

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The Leukocyte Activation Cocktail is a laboratory reagent used to activate and stimulate white blood cells (leukocytes) in vitro. It contains a combination of chemical compounds that work together to trigger the activation and proliferation of leukocytes, which are crucial for immune response and research.

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Lab products found in correlation

Golgiplug, by BD (19 mentions) Cytofix cytoperm kit, by BD (12 mentions) Fixation permeabilization kit, by Thermo Fisher Scientific (10 mentions) Intrapre reagent, by Beckman Coulter (9 mentions) Cytofix cytoperm, by BD (8 mentions) Facscanto 2 system, by BD (7 mentions) Facscanto 2, by BD (6 mentions) Facscalibur, by BD (6 mentions) Gallios instrument, by Beckman Coulter (5 mentions) Gallios flow cytometer, by Beckman Coulter (5 mentions) Golgistop, by BD (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Lsrfortessa, by BD (4 mentions) Fixation permeabilization solution kit, by BD (4 mentions) Ionomycin, by BD (4 mentions) Fixation permeabilization kit, by BD (4 mentions) Cytofix cytoperm soln kit, by BD (4 mentions) Facsverse flow cytometer, by BD (4 mentions) Cytofix cytoperm fixation permeabilization solution kit, by BD (4 mentions) Ifn γ, by BD (4 mentions)

185 protocols using leukocyte activation cocktail

1

Comprehensive Treg Cell Phenotyping

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Treg cells were identified as anti-CD4-positive and anti-CD25-positive. Where indicated, additional markers were evaluated using anti-Human Ki-67 PerCP-eFluor® 710, human Neuropilin-1 PerCP MAb, anti-Human CD152 (CTLA-4) PE, HU FOXP3 APC, and Gata3 PE. Before intracellular staining, Cytofix/Cytoperm Kit and Pharmingen Leukocyte Activation Cocktail with BD GolgiPlug were used for cell fixation and permeation.
PE Annexin V Apoptosis Detection Kit I was used to identify the apoptosis of Treg cells. For intracellular cytokine production, PBMCs were stimulated with Leukocyte Activation Cocktail with BD GolgiPlug for 3 hours before staining. Cytofix/Cytoperm Kit and Pharmingen Leukocyte Activation Cocktail with BD GolgiPlug were used for intracellular cytokine production. Intracellular staining was performed using APC Rat anti-Human IL-4, PE Rat anti-Human IL-5, APC Rat anti-Human IL-13, PE Mouse anti-Human IFN-γ, APC Rat anti-Human IL-10, and PE Mouse anti-Human TGF-β1.
Samples were detected with a FACS flow cytometer, and acquired data were analyzed with FlowJo software.
Chen T., Hou X., Ni Y., Du W., Han H., Yu Y, & Shi G. (2018). The Imbalance of FOXP3/GATA3 in Regulatory T Cells from the Peripheral Blood of Asthmatic Patients. Journal of Immunology Research, 2018, 3096183.
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2

Multiparameter Flow Cytometry of KEAP1-Edited T Cells

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Fluorochrome conjugated antibodies to following human antigens were used for flow cytometric analysis of KEAP1 edited cells: TCR-BV421(BioLegend, San Diego, CA), CD4-PerCP-Cy5.5 (BD Biosciences, Franklin Lakes, NJ), CD8-APC (BioLegend, San Diego, CA), CD25-BV605 (eBioscience, San Diego, CA), FoxP3-APC (eBioscience, San Diego, CA), or Alexa488 (BioLegend, San Diego, CA) CD69-APC-Cy7 (BD Biosciences, Franklin Lakes, NJ), IFNγ-PE (BD Biosciences, Franklin Lakes, NJ), TNFα-FITC (BD Biosciences, Franklin Lakes, NJ), IL4-AlexaFluor 488 (BioLegend, San Diego, CA) IL-10-PE or APC (eBioscience, San Diego, CA), and IL17-BV421 or PE (BioLegend, San Diego, CA). T lymphocytes (~5×105) were stimulated with leukocyte activation cocktail (BD Pharmigen, San Jose, CA) containing PMA (Phorbol 12-Myristate 13-Acetate), ionomycin and brefeldin A before staining for surface markers and intracellular cytokines. Labelled samples were analyzed with LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ). Unstained and unstimulated samples were used to correctly identify and gate cell populations during analysis using FlowJo software (Tree Star Inc., Ashland, OR).
Noel S., Lee S.A., Sadasivam M., Hamad A.R, & Rabb H. (2018). KEAP1 editing using CRISPR/Cas9 for therapeutic NRF2 activation in primary human T lymphocytes. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1929-1936.
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3

Multiparametric Analysis of Cytokine Responses

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5–10×106 cells isolated from spleens, MLN, LP or IEL compartments were incubated with 1 μg/ml Golgiplug (BD Biosciences, San Jose, CA) with or without 10 μg/ml LLO-derived peptide NEKYAQAYPNVS (LLO190–201) or with leukocyte activation cocktail (BD Pharmigen, San Jose, CA) for 5 h at 37°C and 5% CO2. IELs were also incubated with congenic mismatched, naïve splenocytes. Cells were then stained for surface markers for 30 min at 4°C. Cells were treated with BD Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C prior to intracellular staining with anti-IFNγ, anti-IL-2, anti-IL-17A, and anti-TNFα.
Romagnoli P., Fu H., Qiu Z., Khairallah C., Pham Q., Puddington L., Khanna K., Lefrançois L, & Sheridan B. (2016). Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection. Mucosal immunology, 10(2), 520-530.
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4

Multiparametric Analysis of Cytokine Responses

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5–10×106 cells isolated from spleens, MLN, LP or IEL compartments were incubated with 1 μg/ml Golgiplug (BD Biosciences, San Jose, CA) with or without 10 μg/ml LLO-derived peptide NEKYAQAYPNVS (LLO190–201) or with leukocyte activation cocktail (BD Pharmigen, San Jose, CA) for 5 h at 37°C and 5% CO2. IELs were also incubated with congenic mismatched, naïve splenocytes. Cells were then stained for surface markers for 30 min at 4°C. Cells were treated with BD Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C prior to intracellular staining with anti-IFNγ, anti-IL-2, anti-IL-17A, and anti-TNFα.
Romagnoli P., Fu H., Qiu Z., Khairallah C., Pham Q., Puddington L., Khanna K., Lefrançois L, & Sheridan B. (2016). Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection. Mucosal immunology, 10(2), 520-530.
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5

Multiparameter Flow Cytometry Analysis

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Cells were stained with the antibodies listed in Supporting Information Additional Table S2, live/dead dye (Thermo Fisher Scientific) and anti-CD16/CD32 (Bio X Cell) for 20 min at 4 °C in the dark. All samples were fixed with 2% paraformaldehyde for 20 min. For detection of Vγ4+ cells, 20 µg of 1C10-1F7 antibody was used to stain the cells prior to secondary staining with a polyclonal rat anti-mouse IgG (Invitrogen). Cells were then stained with the other conjugated antibodies. For functional analysis, MLN cells were cultured at 37 °C, 5% CO2 for 4 h with BD Leukocyte Activation Cocktail (BD Pharmingen) in IMDM media containing 10% FBS, 10mM HEPES, 1mM sodium pyruvate, 2mM GlutaMAX™ supplement and 1X MEM non-essential amino acids solution (Thermo Fisher Scientific). Intracellular staining was performed using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer’s instructions. Stained cells were acquired on a LSRFortessa (BD Biosciences). Data were analyzed with FlowJo software (TreeStar).
Khairallah C., Chu T.H., Qiu Z., Imperato J.N., Yang D, & Sheridan B.S. (2022). The accumulation of Vγ4 T cells with aging is associated with an increased adaptive Vγ4 T cell response after foodborne Listeria monocytogenes infection of mice. Immunity & Ageing : I & A, 19, 19.
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6

Multiparameter Analysis of Dendritic and T Cells

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Anti-mouse (CD3, CD11b, CD19, IgM, CD49b, Ter119) PE or CD11b PE-CY7, CD11c FITC or APC-CY7, B220 PERCP-CY5.5 and PDCA1 ef450 were used for pDC analysis and purchased from BD Bioscience (San Jose, CA), Biolegend (San Diego, CA), and eBioscience (San Diego, CA). Stimulation of pDC for cytokine profile analysis was done using 50μM of CpG (ODN 1585, Invivogen, San Diego, CA) of whole bone marrow in complete RPMI 1640 supplemented with 10% FBS, 100 U/mL of penicillin, 100μg/mL of streptomycin, and 50μM each of 2-mercaptoethonaol, nonessential amino acids, HEPES, and sodium pyruvate (complete media) in 10cm wells for 9 hours at 37°C. BD golgiplug was added at hour 3. Intracellular analysis of pDC was done using BD Bioscience cytofix/cytoperm kit and anti-mouse IDO PerCP-Cy5.5, IFNα FITC, IL-10 PECY7 and IL-12 APC antibodies.
Splenocytes were stained using anti-mouse CD3 FITC, CD4 PE-CF594, CD8 PERCP-CY5.5, and CD25 APC-CY7. T cells were stimulated with BD leukocyte activation cocktail and golgiplug for 6 hours. Intranuclear staining was done using the eBioscience fixation kit and Tbet PE-CY7, GATA3 PE, RORγT APC, and FoxP3 PE antibodies. Data were acquired with a FACS Aria (BD, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, Oregon).
Hassan M., Ulezko A., Ming Li J., Hosoba S., Rupji M., Kowalski J., Perricone A.J., Jaye D.L., Marsh H., Yellin M., Devine S, & Waller E.K. (2018). Flt3L treatment of bone marrow donors increases graft plasmacytoid dendritic cell content and improves allogeneic transplant outcomes. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 25(6), 1075-1084.
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7

Intracellular Cytokine Staining and FACS Analysis

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After blocking FcR, cells were incubated with appropriately diluted antibodies. Acquisition was performed using a SLRII (BD Biosciences, Mountain View, CA) and data analysis was conducted using FlowJo software (Tree Star Inc., Ashland, OR). For intracellular cytokines staining, cells were re-stimulated with BD Leukocyte Activation Cocktail for 4 h. FACS analysis was gated on the live cells only by using LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen Life Technologies). FACS analysis of TILs was gated on live CD45+TCRβ+(or CD3+) cells.
Chen X., Yang Y., Zhou Q., Weiss J.M., Howard O.Z., McPherson J.M., Wakefield L.M, & Oppenheim J.J. (2014). Effective Chemoimmunotherapy with Anti-TGFβ Antibody and Cyclophosphamide in a Mouse Model of Breast Cancer. PLoS ONE, 9(1), e85398.
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8

Quantification of IL-17A and RORγt Levels

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Spleens were ground and filtered through a 70 μm cell strainer. RBCs were lysed using RBC lysis buffer (eBioscience). Isolation of renal leukocytes was processed as discussed in the previous step. Approximately 1x106 of either spleen of kidney cells were resuspended in RPMI medium supplemented with 5% FBS and stimulated with 2 µl of BD Leukocyte Activation Cocktail (ionomycin and phorbol 12-myristate 13-acetate (PMA) along with the Golgi inhibitor, brefeldin A) at 37°C for 5 hrs. Cells were washed and stained first with LIVE/DEAD® Fixable Violet dead cell stain (Invitrogen, Carlsbad, CA). The following surface antibodies were then added for 30 minutes: BV510-conjugated anti-CD45 antibody (clone 30-F11), PerCP-Cy5.5-conjugated anti-CD3 antibody (BioLegend, clone 17A2), and APC-H7-conjugated anti-CD4 antibody (BD Biosciences, clone GK1.5). Intracellular staining was then performed with the BD Cytofix/CytopermTM Plus fixation/permeabilization solution kit following the manufacturer's instructions (BD Biosciences) and using phycoerythrin (PE)-conjugated anti-IL-17A antibody (eBioscience, clone eBio17B7). We followed exactly the same protocol to detect RORγt in blood and gastric tissues, however, we did not stimulate with ionomycin/PMA. PE-conjugated anti-RORγt antibody (eBioscience, clone B2D) was used.
Soutto M., Saleh M., Arredouani M.S., Piazuelo B., Belkhiri A, & El-Rifai W. (2017). Loss of Tff1 Promotes Pro-Inflammatory Phenotype with Increase in the Levels of RORγt+ T Lymphocytes and Il-17 in Mouse Gastric Neoplasia. Journal of Cancer, 8(13), 2424-2435.
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9

Quantifying Human Th17 Cell Proportion

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To determine the proportion of Th17 cells, aliquots of PBMCs were incubated for 4 h with 4 μl of BD Leukocyte Activation Cocktail containing BD GolgiPlug (BD Biosciences, Franklin Lakes,) in an incubator at 37℃. Surface staining was performed using an Alexa Fluor®488‐conjugated mouse anti‐human CD4 antibody (clone RPA‐T4; BD Biosciences), phycoerythrin‐conjugated mouse anti‐human CD25 antibody (clone M‐A251; BD Biosciences). Cells were incubated with the antibodies for 20 min in the dark and then resuspended in BD Transcription Factor Buffer (BD Biosciences). Intracellular staining was performed using the Brilliant Violet 421‐conjugated mouse anti‐human IL‐17 antibody (clone N49‐653; BD Biosciences) or an isotype control. Flow cytometric analysis was performed to analyze CD25+IL‐17+ T cells using a CD4+ gate, using a previously described method with some modifications.20 The purity of the isolated Th17 cells was determined using the same procedure described above.
Han L., Lv Q., Guo K., Li L., Zhang H, & Bian H. (2022). Th17 cell‐derived miR‐155‐5p modulates interleukin‐17 and suppressor of cytokines signaling 1 expression during the progression of systemic sclerosis. Journal of Clinical Laboratory Analysis, 36(6), e24489.
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10

BALF Cytokine Profiling by Flow Cytometry

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BALF was harvested in PBS as described above. All collected cells were pelleted and resuspended in 100 μl of RPMI containing 10% fetal calf serum (FCS), penicillin-streptomycin (2,200 U/ml; Gibco), and a gentamicin sulfate solution (1 mg/ml). BALF cells were then plated in a 96-well round-bottom plate and restimulated with BD Leukocyte Activation Cocktail containing BD GolgiPlug (BD Biosciences). Stimulations were done in accordance with the manufacturer’s instructions. Six hours after activation, BALF cells were surface stained with fluorescently labeled antibodies to Thy1.2, CD4, and CD8. Samples were fixed in 1% paraformaldehyde overnight. Prior to intracellular staining, the samples were permeabilized with 1× BD-Perm/Wash buffer in accordance with the manufacturer’s instructions. Intracellular cytokine staining was done with fluorescently labeled antibodies to IFN-γ, IL-17A, and tumor necrosis factor alpha (TNF-α) diluted in 1× BD-Perm/Wash for 30 min on ice. Samples were immediately washed and analyzed by flow cytometry as described below.
Masso-Silva J., Espinosa V., Liu T.B., Wang Y., Xue C, & Rivera A. (2018). The F-Box Protein Fbp1 Shapes the Immunogenic Potential of Cryptococcus neoformans. mBio, 9(1), e01828-17.
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