Staphylococcal enterotoxin e, by Toxin Technology - Product details

1

T Cell-B Cell Conjugation Assay

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Jurkat T cells and Nalm6 B cells (DSMZ, negative for Mycoplasma) were labeled with 0.25 µM CMFDA and 1 µM CMTMR, respectively, for 20 min in PBS at 37°C. Jurkats (0.4x10⁵ cells) were mixed with 3x10⁵ Nalm6 cells in the absence or presence of staphylococcal enterotoxin E (Toxin Technology Inc.) in a total volume of 20 µl RPMI in 96 well v-bottom plates. After 20 min conjugation was stopped by adding 80 µl of 4% PFA. Samples were analyzed by flow cytometry. Percentage of conjugates was calculated by dividing the number double positive conjugates by the total number of Jurkat cells.
Mouse T cells: 2x10⁵ CD44^lowCD4⁺ T cells purified from Wnk1^+/+dLck-Cre or Wnk1^fl/fldLck-Cre mice were mixed with 2x10⁵ CH27 B cells in the absence or presence of 1 µg/ml staphylococcal enterotoxin B (Sigma) in a total volume of 20 µl RPMI in 96 well V-bottom plates. After 20 min conjugation was stopped by adding 80 µl of 4% PFA and cells were stained with antibodies against CD4, CD44, Vβ8, CD19 and B220 and analyzed by flow cytometry. Percentage of conjugates was calculated by dividing the number of conjugates (Vβ8⁺CD19⁺B220⁺) by the total number of Vβ8⁺ cells.

Köchl R., Thelen F., Vanes L., Brazao T.F., Fountain K., Xie J., Huang C.L., Lyck R., Stein J.V, & Tybulewicz V.L. (2016). WNK1 kinase balances T cell adhesion versus migration in vivo. Nature immunology, 17(9), 1075-1083.

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T Cell-B Cell Conjugation Assay

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Jurkat T cells and Nalm6 B cells (DSMZ, negative for Mycoplasma) were labeled with 0.25 µM CMFDA and 1 µM CMTMR, respectively, for 20 min in PBS at 37°C. Jurkats (0.4x10⁵ cells) were mixed with 3x10⁵ Nalm6 cells in the absence or presence of staphylococcal enterotoxin E (Toxin Technology Inc.) in a total volume of 20 µl RPMI in 96 well v-bottom plates. After 20 min conjugation was stopped by adding 80 µl of 4% PFA. Samples were analyzed by flow cytometry. Percentage of conjugates was calculated by dividing the number double positive conjugates by the total number of Jurkat cells.
Mouse T cells: 2x10⁵ CD44^lowCD4⁺ T cells purified from Wnk1^+/+dLck-Cre or Wnk1^fl/fldLck-Cre mice were mixed with 2x10⁵ CH27 B cells in the absence or presence of 1 µg/ml staphylococcal enterotoxin B (Sigma) in a total volume of 20 µl RPMI in 96 well V-bottom plates. After 20 min conjugation was stopped by adding 80 µl of 4% PFA and cells were stained with antibodies against CD4, CD44, Vβ8, CD19 and B220 and analyzed by flow cytometry. Percentage of conjugates was calculated by dividing the number of conjugates (Vβ8⁺CD19⁺B220⁺) by the total number of Vβ8⁺ cells.

Köchl R., Thelen F., Vanes L., Brazao T.F., Fountain K., Xie J., Huang C.L., Lyck R., Stein J.V, & Tybulewicz V.L. (2016). WNK1 kinase balances T cell adhesion versus migration in vivo. Nature immunology, 17(9), 1075-1083.

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NF-κB Activation in Jurkat Cells

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NF-κB GFP reporter Jurkat cells were resuspended in RPMI-1640 media with 0% FBS and were serum starved for 4 h. After starvation, cells were counted and 5 × 10⁵ cells were plated in a V-bottom 96-well plate. Cells were stimulated with 1 μg/ml αCD3 plus 0.5 or 1 μg/ml αCD28, and returned to the incubator at 37°C, 5% CO₂, for 16 h. In experiments analyzing the effect of anti-TIM-3 (F38.2E2), cells were pre-treated with anti-TIM-3 for 1 h before stimulation with αCD3/αCD28. In experiments using Raji B cell-based stimulation, Raji B cells were loaded with 30 ng/ml Staphylococcal enterotoxin E (Toxin Technology Inc., catalog #ET404) for 30 min at 37°C and were washed in serum-free RPMI-1640 media to remove excess toxin. Jurkat NF-κB GFP reporter cells were serum starved in RPMI-1640 media with 0% FBS for 3 h. After starvation, 2 × 10⁵ Jurkat cells were mixed with 10⁵ SEE-loaded Raji B cells per well in a 96-well U-bottom plate, as described [43 (link)]. The plate was centrifuged for 1 min at 300×g to initiate cell contact, and the plate was returned to the incubator at 37°C, 5% CO₂, for 6 h. Following antibody or cell-based stimulation, medium was collected for analysis of IL-2 secretion by ELISA (R & D Systems, cat #D2050), following the IL-2 Quantikine ELISA manual. Experiments were performed in technical duplicate and biological triplicate.

Smith C.M., Li A., Krishnamurthy N, & Lemmon M.A. (2021). Phosphatidylserine binding directly regulates TIM-3 function. Biochemical Journal, 478(17), 3331-3349.

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T Cell Activation Assay with CADA

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Jurkat or naive CD4⁺ T cells were plated at 2.8 x 10⁵ cells/mL in Falcon round-bottom 96-well plates in presence or absence of 10 μM of CADA. After 48h, T cells were activated by adding Staphylococcal enterotoxin E (Toxin Technology) or Staphylococcal enterotoxin B (Sigma-Aldrich)-stimulated Raji-GFP cells at a concentration of 1.2 x 10⁶ cells/mL. Raji cells were labeled with GFP to distinguish them from Jurkat and naive CD4⁺ T cells by flow cytometry. Expression of the early activation marker CD69 was detected by flow cytometry 24h later. In parallel, proliferation of naive CD4⁺ T cells was quantified by [³H]-thymidine incorporation (see protocol MLR below). For these experiments, after the loading of the Raji-GFP cells with the superantigen, Raji-GFP cells were incubated with mitomycin C (Sigma-Aldrich) to prevent their proliferation.

Claeys E., Pauwels E., Humblet-Baron S., Provinciael B., Schols D., Waer M., Sprangers B, & Vermeire K. (2021). Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes. Frontiers in Immunology, 12, 650731.

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5

Cytokine Release Assay for Bispecific Antibodies

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DCs (1 × 10⁴) and CD4+ T cells (1 × 10⁵) were seeded in RPMI medium containing 0.1 ng/mL staphylococcal enterotoxin E (Toxin Technology, USA) in a 96-well plate and incubated with serial five-fold dilutions of IBI323, Bi127, IBI110, and IgG (with a starting concentration of 200 nM). Four days later, the concentration of INF-γ and interleukin (IL)-2 in culture supernatant was measured using a Cisbio (Bedford, USA) kit.

Jiang H., Ni H., Zhang P., Guo X., Wu M., Shen H., Wang J., Wu W., Wu Z., Ding J., Tang R., Zhou S., Chen B., Yu M., Jing H, & Liu J. (2021). PD-L1/LAG-3 bispecific antibody enhances tumor-specific immunity. Oncoimmunology, 10(1), 1943180.

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6

T Cell Activation Signaling Assay

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MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) powder, CMFDA (5-chloromethylfluorescein diacetate) and CMRA (5-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) cell staining dyes, was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-CD3 antibodies and anti-CD28 antibodies were purchased from Bioxcell (West Lebanon, NH, USA). Human IL-2 DuoSet^® ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). Staphylococcal enterotoxin E (SEE) was purchased from Toxin Technology (Sarasota, FL, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit and ECL Western blotting detection reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD40L antibodies conjugated with APC was purchased from eBiosciences (San Diego, CA, USA). Anti-CD40L neutralizing antibodies were obtained from InvivoGen (San Diego, CA, USA). Anti-CD40L for western blot and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p65, anti-PARP, anti-IκBα, anti-phosphorylated IκBα (S32), anti-phosphorylated ERK (T202/Y204), anti-ERK, anti-phosphorylated p38 (T180/Y182), anti-p38, anti-phosphorylated JNK (T183/Y185), anti-JNK, anti-phosphorylated c-Jun (S73) and anti-c-Jun antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Lee H.S, & Jeong G.S. (2020). Chrysophanol Mitigates T Cell Activation by Regulating the Expression of CD40 Ligand in Activated T Cells. International Journal of Molecular Sciences, 21(17), 6122.

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7

Visualization of TMEM16F Localization

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Raji cells were incubated for 2 h with 1 µg/ml of staphylococcal enterotoxin E (SEE; Toxin Technology) at 37°C. After extensive washing, 10⁵ Raji cells were mixed with 10⁵ Jurkat cells and plated onto poly-l-lysine-coated coverslips in 24-well plates, incubated for 30 min at 37°C, and then fixed with 2% paraformaldehyde (PFA). To determine the localization of TMEM16F, reporter (TMEM16F-RFP) or TCR-β were used to distinguish Jurkat from Raji cells. For immunofluorescence assays, samples were permeabilized with 0.1% Triton X-100, blocked with 10% FBS plus 2% goat serum in PBS, and stained for TCR-Vβ8 (cat. no. 555604; BD), followed by Alexa Fluor 555–conjugated goat anti–mouse IgG2b secondary antibody (A-21147; Life Technologies). All samples were mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). Images were taken using a FV1000 confocal microscope with a water objective lens (UPlanAPO 60×; NA 1.20; Olympus). For 3D and z-axis image reconstruction, 20 confocal sections, 0.4 µm apart, were assembled using ImageJ/Fiji software (NIH).

Hu Y., Kim J.H., He K., Wan Q., Kim J., Flach M., Kirchhausen T., Vortkamp A, & Winau F. (2016). Scramblase TMEM16F terminates T cell receptor signaling to restrict T cell exhaustion. The Journal of Experimental Medicine, 213(12), 2759-2772.

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Staphylococcal enterotoxin e

Lab products found in correlation

7 protocols using staphylococcal enterotoxin e

T Cell-B Cell Conjugation Assay

T Cell-B Cell Conjugation Assay

NF-κB Activation in Jurkat Cells

T Cell Activation Assay with CADA

Cytokine Release Assay for Bispecific Antibodies

T Cell Activation Signaling Assay

Visualization of TMEM16F Localization

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