Cell activation cocktail without brefeldin a

Manufactured by BioLegend

Sourced in United States

The Cell Activation Cocktail (without Brefeldin A) is a laboratory reagent designed to stimulate the activation of cells. It contains a proprietary blend of cytokines and other molecules that promote cellular activation. The core function of this product is to induce activation in cell cultures, enabling further study and analysis. No additional interpretation or extrapolation is provided.

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Close spelling variants of this product

Brefeldin A (659 citations) Cell Activation Cocktail (119 citations) Activation cocktail (5 citations)

8 protocols using cell activation cocktail without brefeldin a

Isolation of Activated Immune Cell Subsets

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Cells were collected for analysis 6 days after electroporation, and
10–20 × 10⁶ cells were portioned off and sorted based
on GFP expression only. The remaining cells were sorted based on GFP positivity,
as well as target expression using an APC fluorescent antibody targeting either
IL2RA (CD25) (Tonbo, catalog no. 20–0259-T100), IL-2 (Biolegend, catalog
no. 500310) or CTLA4 (Biolegend, catalog no. 349908). All antibodies were used
at a 1:25 dilution for staining. Cells sorted for IL2RA underwent surface
staining according to the manufacturer’s protocol. Cells sorted for IL-2
were treated with Cell Activation Cocktail with Brefeldin A (Biolegend, catalog
no. 423304) for 4 h before fixation, and were fixed using the BD
Cytofix/Cytoperm kit (Becton Dickinson, catalog no. 554714) according to the
manufacturer’s protocol. Cells sorted for CTLA4 were treated with Cell
Activation Cocktail without Brefeldin A (Biolegend, catalog no. 423302) for 4 h
before fixation, and were fixed using the Foxp3 Fix/Perm buffer set (Biolegend,
catalog no. 421403) according to the manufacturer’s protocol. Cells were
sorted using a BD FACS Aria II and FACSDiva v.8.0.1.

Freimer J.W., Shaked O., Naqvi S., Sinnott-Armstrong N., Kathiria A., Garrido C.M., Chen A.F., Cortez J.T., Greenleaf W.J., Pritchard J.K, & Marson A. (2022). Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks. Nature genetics, 54(8), 1133-1144.

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Lymphocyte Activation and PBMC Isolation

Cited 1 time

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Single cell suspensions of lymphocytes were prepared from the spleen of sacrificed mice. Red blood cell lysis was performed with ACK Lysis Buffer (Quality Biologicals, MD, USA).(26 (link)) Human blood samples (buffy coats) were obtained from the National Institutes of Health Blood Research Services for peripheral blood mononuclear cell (PBMC) isolation and prepared as previously described.(27 (link)) Fresh PBMCs were isolated by Ficoll density gradient centrifugation (Biochrom, Berlin, Germany). Splenocyte or PBMC cell activation was performed with the Cell Activation Cocktail without Brefeldin A (BioLegend) according to the manufacturer’s protocol. Briefly, splenocytes or PBMCs were cultured in 1mL medium with 2μL of the Cell Activation Cocktail for 24 hours. Cells and supernatant were then transferred from the six well plate into an Eppendorf tube which was centrifuged and supernatant collected for coculture with tumor cells.

Brown Z.J., Yu S.J., Heinrich B., Ma C., Fu Q., Sandhu M., Agdashian D., Zhang Q., Korangy F, & Greten T.F. (2018). Indoleamine 2,3-dioxygenase provides adaptive resistance to immune checkpoint inhibitors in hepatocellular carcinoma. Cancer immunology, immunotherapy : CII, 67(8), 1305-1315.

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Flow Cytometry Analysis of Protein Knockout

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Protein knockout was evaluated by flow cytometry 5–6 d post-editing. T cells were stained with fluorophore-conjugated antibodies for TCRα/β (Biolegend, 306718), β2M (Biolegend, 316304) and PD-1 (Biolegend, 367422) via 1:33 dilution in standard PBS. For PD-1 analysis by flow cytometry, T cells were treated with Cell Activation Cocktail (without brefeldin A) (Biolegend) overnight before staining. Events were collected using a MACSQuant Analyzer 16 (Miltenyi). Data were analyzed using the FlowJo software (v10.8.1)

Lam D.K., Feliciano P.R., Arif A., Bohnuud T., Fernandez T.P., Gehrke J.M., Grayson P., Lee K.D., Ortega M.A., Sawyer C., Schwaegerle N.D., Peraro L., Young L., Lee S.J., Ciaramella G, & Gaudelli N.M. (2023). Improved cytosine base editors generated from TadA variants. Nature Biotechnology, 41(5), 686-697.

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Quantifying RBD-specific T cell Response

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To detect RBD-specific T lymphocyte response, an IFN-γ -based ELISPOT assay was performed. Mice spleens were collected and lymphocytes were isolated. 96-well plates were precoated with anti-mouse IFN-γ antibody overnight at 4°C and then blocked for 2 hours at room temperature. Different concentrations of the recombinant RBD protein were added to the well, and then lymphocytes were added to the plate (1.5 × 10⁵/well). Cell Activation Cocktail (without Brefeldin A) [BioLegend (Cat. No. 423301)] was added as a positive control and cells stimulated with 0.9% NaCl were employed as a negative control. After 24 hours of incubation, the cells were removed, and IFN-γ was captured by biotinylated detection antibody, streptavidin-HRP conjugate and AEC substrate.

Zhou Y., Qu J., Sun X., Yue Z., Liu Y., Zhao K., Yang F., Feng J., Pan X., Jin Y., Cheng Z., Yang L., Ha U.H., Wu W., Li L, & Bai F. (2023). Delivery of spike-RBD by bacterial type three secretion system for SARS-CoV-2 vaccine development. Frontiers in Immunology, 14, 1129705.

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Multiparameter Flow Cytometry Analysis of Activated T Cells

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On day 14 post expansion, 1 × 10⁶ engineered T cells were stimulated with Cell Activation Cocktail (without Brefeldin A) (BioLegend, 423301) or Dynabeads Human T-Activator CD3/CD28 in 96-well round-bottom plates for 5 h at 37 °C. After 1 h of incubation, a mixture of GolgiStop (BD, 554724) and GolgiPlug (BD, 555029) was added. To detect T cell degranulation, mouse anti-human PE-Cy7 CD107a antibody (BD, 561348) or PE-Cy7 isotype (Mouse IgG1κ, BD, 557872) (1 µL/well) were treated during the culture. The cells were then stained with mouse anti-human FITC CD3 (BD, 561807), APC-Cy7 HLA-ABC (BioLegend, 311426), PE HLA-DRDPDQ (Miltenyi Biotec, 130–104-827) and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, L34957) for dead cell exclusion. Using a Fixation/Permeabilization Solution Kit (BD, 554714), the cells were intracellularly stained with BV421 IFN-γ (BD, 562988) and Alx647 TNF-α (BioLegend, 502916) antibodies and analyzed by flow cytometry.

Lee J., Sheen J.H., Lim O., Lee Y., Ryu J., Shin D., Kim Y.Y, & Kim M. (2020). Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy. Scientific Reports, 10, 17753.

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Comprehensive Immunology Research Protocol

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All fluorescently tagged antibodies, αCD28/CD3 antibodies, ELISA kits, RBC lysis buffer, Foxp3/Transcription Factor Staining Buffer Set, EDTA, and Cell Proliferation Dye eFluor^™ 670 were purchased from Invitrogen. All inhibitors (modulators) were purchased from MedChemExpress. Bovine serum albumin (BSA) was purchased from VWR Life Science. HBSS, D PBS, PBS, DMEM, RPMI 1640, AIM-V medium, FBS, HI-FBS, HEPES, and non-essential amino acid solutions were purchased from Gibco. RAW-Dual cells, RAW Blue cells, QB buffer and reagent, EndoFit Ovalbumin (OVA), ODN 1826 (CpG), and all other TLR agonists were purchased from InvivoGen. Spleen Dissociation Medium and EasySep Mouse T-Cell Isolation Kit were purchased from STEMCELL Technologies. β-Mercaptoethanol was purchased from MP Biomedicals. Cell Activation Cocktail (without Brefeldin A), Recombinant Mouse GM-CSF (carrier-free) (20 ng/mL), and LEGENDplex MU Th1/Th2 Panel (8-plex) Kit were purchased from BioLegend. ProT2 MHC Class II tetramers and Pro5 MHC Class I pentamers were purchased from ProImmune. All plates, unless noted otherwise, were purchased from Thermo Fisher Scientific.

Jia S., Kim J., Esser-Kahn A.P, & Deak P. (2024). High-throughput screening identification of novel immunomodulatory combinations for the generation of tolerogenic dendritic cells. Frontiers in Medicine, 10, 1298424.

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Evaluating SARS-CoV-2 Spike-Specific Immunity

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To evaluate cell-mediated immune response, spleen tissues were collected from Balb/c mice that received mRNA-LNPs containing either 1 or 10 µg of Spike encoding mRNA at day 28 post booster immunization. Mouse uncoated ELISA kits (Invitrogen, Waltham, MA, USA) were used upon stimulation of elicited cells with peptide pool comprising SARS-CoV-2 Spike RBD to quantify interferon γ (IFN-γ) and interleukin-4 (IL-4) secretions. Cell activation cocktail (without Brefeldin A, BioLegend, San Diego, CA, USA) and RPMI 1640 medium were employed as the positive and negative controls, respectively.

Naderi Sohi A., Kiani J., Arefian E., Khosrojerdi A., Fekrirad Z., Ghaemi S., Zim M.K., Jalili A., Bostanshirin N, & Soleimani M. (2021). Development of an mRNA-LNP Vaccine against SARS-CoV-2: Evaluation of Immune Response in Mouse and Rhesus Macaque. Vaccines, 9(9), 1007.

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Investigating Methotrexate and NLRP3 Inflammasome

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Methotrexate hydrate (Cas: 59-05-2) was purchased from J&K Scientific, and MCC950 sodium (Cas: 256373-96-3) from Med Chem Express. All anti-mouse antibodies CD80-FITC (104706), CD86-PE (105008), CD11c-APC (117310), CD40-PE (124610), I-A/I-E-PerCP/Cyanine 5.5 (107626), CD3-APC (100312), CD4-FITC (100510), CD8a-PE (100708) and IFN-γ-APC (505810) were procured from BioLegend. Cell Activation Cocktail (without Brefeldin A; 423301), PMA (20 ng/mL), ionomycin (1 µg/mL), Brefeldin A solution (1000×; 420601), and MojoSort^™ Mouse CD8 T Cell Isolation Kit (480008) were also purchased from BioLegend. Rabbit mAbs against caspase-1 (D7F10; #3866), cleaved caspase-1 (Asp297) (D57A2; #4199), IL-1β (D6D6T; #31202), cleaved IL-1β (Asp116) (E7V2A, #63124), and NLRP3 (D4D8T; #15101) were purchased from Cell Signaling. Recombinant mouse IL-4 and GM-CSF were obtained from PeproTech, LPS (L2880) and lipopolysaccharides from Escherichia coli O55:B5 were purchased from Sigma, Imject Alum (77161) was obtained from Thermo Fisher Scientific, nigericin (Cas: 28380-24-7) was purchased from Med Chem Express. To rule out the possibility of endotoxin contamination during MTX preparation, the Toxin Sensor^™ Chromogenic LAL Endotoxin Assay Kit (GenScript, NJ, USA) was used to determine the LPS content. The level of endotoxin in the MTX preparation was lower than 0.1 endotoxin unit/mL.

Shi G.N., Hu M., Chen C., Fu J., Shao S., Zhou Y., Wu L, & Zhang T. (2021). Methotrexate enhances antigen presentation and maturation of tumour antigen-loaded dendritic cells through NLRP3 inflammasome activation: a strategy for dendritic cell-based cancer vaccine. Therapeutic Advances in Medical Oncology, 13, 1758835920987056.

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