Cell stimulation cocktail

Manufactured by Cytek Biosciences

Sourced in United States

The Cell Stimulation Cocktail is a laboratory reagent designed to activate and stimulate cells. It contains a combination of chemical agents that induce cellular responses, including increased cytokine production and cellular activation. The specific composition and concentrations of the ingredients are optimized to effectively stimulate a range of cell types.

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Lab products found in correlation

13 protocols using cell stimulation cocktail

Th17 Cell Differentiation from Naive CD4+ T Cells

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Naïve CD4 cells were isolated from C57BL/6J mouse spleens using immunomagnetic separation beads (Miltenyi Biotec, 130-106-643) according to the manufacturer's instructions and seeded into 96-well plates (5 × 10⁵/well) precoated with anti-CD3 antibody (5 μg/ml) (BioLegend, 100309) with the addition of soluble anti-CD28 antibody (2 μg/ml) (BioLegend, 102102). MSCs, sh-NC-MSCs, sh-Chi3l1-MSCs, or sh-Chi3l1-MSCs plus Stattic (20 μM, Selleck, S7024) were seeded into 96-well plates (5 × 10³/well) 6 h before CD4 cell seeding. Th17 differentiation medium contained TGF-β (1.0 ng/ml) (PeproTech, AF-100-21C), IL-6 (30 ng/ml) (PeproTech, 216-16), IL-1β (20 ng/ml) (PeproTech, 211-11B), IL-23 (20 ng/ml) (BioLegend, 589002), anti-IL-4 (10 μg/ml) (BioLegend, 504102), and anti-IFN-γ (10 μg/ml) (BioLegend, 505833).
Th17 cells were differentiated for 72 h and restimulated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975) for 6 h before further analysis for intracellular cytokines by flow cytometry.

Liu W., Yuan F., Bai H., Liu Y., Li X., Wang Y, & Zhang Y. (2022). hUC-MSCs Attenuate Acute Graft-Versus-Host Disease through Chi3l1 Repression of Th17 Differentiation. Stem Cells International, 2022, 1052166.

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In Vitro Differentiation of Innate Lymphoid Progenitors

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For bulk culture, 1–1.5 × 10⁴ OP9-DL4 stromal cells were pre-seeded in 24-well round bottom plates one night before co-culture. Freshly FACS-sorted Lin^–CD45⁺CD127⁺CD34⁺IL-3RA^+/–progenitors (150 cells) or ILCP (Lin^–CD45⁺CD7⁺CD127⁺CD117⁺) were seeded into the wells with pre-seeded OP9-DL4 stromal cells. For single-cell culture, 3–5 × 10³ OP9-DL4 stromal cells were pre-seeded in 96-well round bottom plates one night before co-culture. Cells of Lin^–CD45⁺CD127⁺CD34⁺IL-3RA⁺ progenitors were sorted in single-cell mode and then plated on the stromal cells. Cells were cultured in Yssel’s medium supplemented with 2% human AB serum (GeminiBio, 100–512) as Dr. James P. Di Santo’s group described in the presence of IL-2, IL-7, IL-1β, IL-23, IL-25 and IL-33 (PeproTECH) for 10–14 days.^{36 (link)} Stromal cells were passaged when the confluence reached about 80%–95%. Co-cultured cells were treated by 0.25% Trypsin-EDTA (Sigma, 59428 C) solution and collected by spinning at 500× g for 5 min at 4 °C. All cultures were analyzed in parallel with negative control wells that contained OP9-DL4 stromal and identical culture medium without progenitors. To detect the cytokine expression, cells were stimulated with cell stimulation cocktail (Tonbo, TNB-4975), 2 μL per sample, for 4 h. Intracellular staining of cytokines was performed with Cytofix/Cytoperm Kit (BD, 554714).

Liu C., Gong Y., Zhang H., Yang H., Zeng Y., Bian Z., Xin Q., Bai Z., Zhang M., He J., Yan J., Zhou J., Li Z., Ni Y., Wen A., Lan Y., Hu H, & Liu B. (2021). Delineating spatiotemporal and hierarchical development of human fetal innate lymphoid cells. Cell Research, 31(10), 1106-1122.

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Cell Stimulation and Antibody Staining

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Samples were stimulated with Cell Stimulation Cocktail (Tonbo Biosciences, catalog TNB-4975) for 4 hours at 37°C, then were stained with antibodies described in Supplemental Table 2. Psoriatic samples were collected contemporaneously with HS samples and have been described in a previous publication (28 (link)).

Lowe M.M., Naik H.B., Clancy S., Pauli M., Smith K.M., Bi Y., Dunstan R., Gudjonsson J.E., Paul M., Harris H., Kim E., Shin U.S., Ahn R., Liao W., Hansen S.L, & Rosenblum M.D. (2020). Immunopathogenesis of hidradenitis suppurativa and response to anti–TNF-α therapy. JCI Insight, 5(19), e139932.

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Splenic T-cell Activation and Cytokine Analysis

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Mouse spleens were dissociated with GentleMACS Octo (Miltenyi-Biotech). T-cells were activated overnight in TexMACS + 10% FBS medium. 2 µL/mL Cell stimulation cocktail (cat#TNB-4970-UL100, Tonbo-Biosciences) was added for 2 h at 37 °C. 1µL/mL Brefeldin A solution (cat# TNB-4506-L001, Tonbo-Biosciences) was then added for 2 h at 37 °C. Extracellular labeling of T-cells was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech), VioGreen anti-CD4 (cat#130-118-693, Miltenyi-Biotech), and APC-Vio770 anti-CD8 (cat#130-120-806, Miltenyi-Biotech) antibodies. After fixation and permeabilization of cells, intracellular labeling of T-cells was performed with APC anti-IFNγ antibody (cat#130-123-283, Miltenyi-Biotech). Samples were analyzed using MacsQuant™ 16 Flow cytometer.

Confino H., Dirbas F.M., Goldshtein M., Yarkoni S., Kalaora R., Hatan M., Puyesky S., Levi Y., Malka L., Johnson M., Chaisson S., Monson J.M., Avniel A., Lisi S., Greenberg D, & Wolf I. (2022). Gaseous nitric oxide tumor ablation induces an anti-tumor abscopal effect. Cancer Cell International, 22, 405.

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Studying Allergic Lung Inflammation

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To increase DC number to sort by FACS, up to 4 WT and 4 Plpp6^-/- Balb/c mice were exposed to 25μg of HDM and 1μg of lipopolysaccharide (LPS) in 25μL every 24h for 3 days. 24h after the last exposure, mice were euthanized, and lungs and MLNs were processed to obtain single cells suspension as described above. DCs were sorted in a BD FACSAria as CD45⁺, CD11c⁺, MHC-II⁺ and autofluorescent FITC (macrophages) were excluded. DCs were cultured for 24h in complete media (described above) with 100 μg/mL of ovalbumin (OVA). For naïve T cells sorting, 2 naïve WT, 2 Plpp6^-/- and 2 C. Cg-Tg (DO11.10) 10 Dlo/J mice were euthanized and the spleens were harvested and homogenized, and RBCs lysed. Cell suspensions were stained with the following mouse antibodies: anti-CD3 (clone 17A2), anti-CD4 (clone RM4-5), anti-CD25 (clone PC 61.5), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14). Naïve T cells were sorted as CD3⁺, SSCA^low, CD4⁺, CD25⁻, CD44^-/low and CD62L^high. T cells were cultured with DCs in a 1:10 (DC:Tcell) ratio for 3 days. On day 3 of the co-incubation, cells were stimulated for 4h with cell stimulation cocktail (Tonbo biosciences, catalog number TNB-4975). Cells were harvested and stained in FoxP3 staining Kit (Thermo Fisher Scientific) with the following mouse antibodies with the same clones as described above: anti-CD45, anti-CD3, anti-CD4 and anti-IL-13.

Brüggemann T.R., Carlo T., Krishnamoorthy N., Duvall M.G., Abdulnour R.E., Nijmeh J., Peh H.Y., Filippakis H., Croze R.H., Goh B., Oh S.F, & Levy B.D. (2022). Mouse phospholipid phosphatase 6 regulates dendritic cell cholesterol, macropinocytosis, and allergen sensitization. iScience, 25(10), 105185.

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Activating Mononuclear Cells for Analysis

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Mononuclear cells (MNCs) were thawed, counted, and resuspended at 1 × 10⁶ cells/ml in RPMI-1640 medium (Euroclone), supplemented with 10% fetal bovine serum (FBS) (Euroclone), 2 mM l-glutamine (Euroclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Euroclone). Cells were exposed for 4 h to the cell stimulation cocktail (Tonbo Biosciences, San Diego, CA, USA), containing brefeldin-A, monensin, ionomycin (IONO), and phorbol-myristate acetate (PMA), at 37°C, 5% CO₂.

La Sala L., Gandini S., Bruno A., Allevi R., Gallazzi M., Senesi P., Palano M.T., Meregalli P., Longhi E., Sommese C., Luzi L, & Trabucchi E. (2022). SARS-CoV-2 Immunization Orchestrates the Amplification of IFNγ-Producing T Cell and NK Cell Persistence. Frontiers in Immunology, 13, 798813.

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Analyzing Tumor-Immune Cell Interactions

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OT-I splenocytes were stimulated with OVA (30 ng/mL) ± fostriecin at 1 and 10 nM. At 72 hours, cells were restimulated with OVA peptide in presence of GolgiPlug and incubated for 4 hours at 37°C in a 5% CO2 incubator. The cells were surface markers (anti- mouse-CD3, CD8, live/dead stain). Cells were fixed, permeabilized and stained for cytokines using an anti-interferon (IFN)-γ and tumor necrosis factor (TNF)-α antibody. For cell co-culture experiments, OVCAR8 cells were treated with fostriecin or transfected with either PPP4C or control or PPP4R3B siRNA, followed by carboplatin treatment (2.5 µM). On day 5, cells were trypsinized and seeded on to a flat bottom 12-well plate. After 6 hours, NK-92 cells were added in a 1:1 (tumor:NK cell) ratio. At the end of 14 hours co-culture, NK-92 cells were collected and restimulated with cell stimulation cocktail (#TNB-4975-UL100, Tonbo Biosciences) for 4 hours and transferred to Laminar Wash 96-well plate (Curiox Biosystems, Seoul, South Korea) for intracellular staining. Cells were washed by laminar flow (8–10 cycles) using the Curiox Laminar Wash System HT1000. All antibodies used in flow cytometry are provided in online supplemental table 4. Flow data analysis was performed with FlowJo (V.10.8.0)

Raja R., Wu C., Bassoy E.Y., Rubino TE J.r., Utagawa E.C., Magtibay P.M., Butler K.A, & Curtis M. (2022). PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling. Journal for Immunotherapy of Cancer, 10(12), e005026.

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Tumor and Lymph Node Single-Cell Isolation

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The tumors and lymph nodes collected from mice were mechanically dissociated. Lymph nodes were incubated in 2 mL tissue dissociation media (RPMI-1640 media; Fisher Healthcare; MT10040CV) supplemented with 0.5% BSA (Sigma-Aldrich; A1470), 0.75 mg/mL collagenase (Type II; Thermo Scientific; 17,101–015), 30 µg/mL DNase I (Sigma-Aldrich; 10104159001) at 180 rpm (shaking) 37°C for 20 min. Tumor tissue dissociation was done with the gentleMACS Octo dissociator with heaters (Miltenyi Biotec) using 2.5 mL tissue dissociation media. Cell suspensions were filtered through 70 micron nylon cell strainer (Fisher Scientific; 22-363-548). For ex vivo restimulation, cells were treated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975-UL400) for 4 hours, followed by cell surface and intracellular staining. Absolute counting beads (Thermo Scientific, C36950) were included to obtain total cell counts in the samples. All antibodies used for flow cytometry are provided in online supplemental table 4.

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Multicolor Flow Cytometry Analysis

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Aliquots of cells were transferred into a sterile 24-well plate with 1 mL of complete ISCOVES culture media supplemented with 2 mM L-glutamine, 5 mM Sodium Pyruvate, non-essential amino acids (MEM NEAA), 10 mM HEPES, 100 U/mL Penicillin, 100 U/mL Streptomycin and 2 × 10⁻⁵M of 2β-Mercaptoethanol. The cells were stimulated in vitro with 1 μL of cell stimulation cocktail (Tonbo Biosciences, San Diego, CA, USA). The plate was then placed into a HERAcell 150i incubator set at 37 °C and 5% CO₂ for 6 h. After incubation, the cells were harvested and spun at 300× g at 4 °C for 5 min.
The cells were washed with FACS buffer then incubated with Fc block in the dark at 4 °C for 20 min. Fluorescent antibodies were used against surface markers to label cells of interest: PE-Cy5-conjugated anti-CD11b (Tonbo 55-0112-U100) and PE-conjugated F4/80 (Biolegend, 123110) at 4 °C in the dark for 40 min. After incubation, cells were fixed with 300 μL of 2% paraformaldehyde. The cells were then permeabilized using permeabilization buffer (Tonbo Biosciences, San Diego, CA, USA) and stained with IFNγ (Biolegend, 505850). The cells were then resuspended with 200 μL of FACS buffer and filtered for analysis on the FC500 flow cytometer.

Murr N.J., Olender T.B., Smith M.R., Smith A.S., Pilotos J., Richard L.B., Mowa C.N, & Opata M.M. (2021). Plasmodium chabaudi Infection Alters Intestinal Morphology and Mucosal Innate Immunity in Moderately Malnourished Mice. Nutrients, 13(3), 913.

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Characterizing Tissue-Resident T Cell Subsets

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Surface staining of live cells were done in the presence of Fc block and fixable viability dye. For intracellular cytokine staining, cells were stimulated with cell stimulation cocktail (Tonbo Biosciences) for 4-5 hours, subjected to surface marker staining in the presence of Fc block, fixed in 4.2% paraformaldehyde, permeabilized and stained with the indicated anti-cytokine antibodies in intracellular perm/wash buffer (BioLegend) for 2 hours at 4 °C. Nuclear staining was performed with Foxp3 staining buffer kit (eBioscience). T cells were identified via lymphocyte/single cells/live cells/TCRβ⁺ gating. CD4⁺Foxp3^RFP+ T cells were defined as Foxp3⁺ gating Treg cells, while CD4⁺Foxp3^RFP- T cells were defined as conventional CD4⁺ T cells. Tissue resident or circulating status was then determined via fluorescent in vivo IV staining of CD45.2 (CD45.2^IV- for tissue resident cells and CD45.2^IV+ for circulating cells respectively). IL-10^GFP was utilized to analyze ex vivo IL-10 production by T cells during influenza infections.

McGee M.C., Zhang T., Magazine N., Islam R., Carossino M, & Huang W. (2022). PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu. Frontiers in Immunology, 13, 984476.

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