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2 protocols using anti tnf α eflour 450 clone mab11

1

Isolation and Cytokine Analysis of CD4+ T Cells

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Peripheral venous blood was obtained from study participants. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by density centrifugation using Biocoll (Merck, Darmstadt, Germany). 106 CD4+ enriched cell fractions isolated by CD4+ T Cell Kit (Miltenyi) were plated onto U-bottom plates and were restimulated for 4 hours at 37°C, 5% CO2 in a humidified incubator. Restimulation was conducted in a final volume of 200 μl RPMI 1640 (Sigma) supplemented with 10% FBS (Merck), 100 U/ml penicillin (Sigma), 100 mg/ml streptomycin (Sigma), 50 ng/ml PMA (Sigma), 250 ng/ml ionomycin (Sigma) and 1.3 μl/ml Golgistop (BD). After restimulation cells were stained with Life/Dead Fixable Aqua Dead cell kit (Thermo Fisher) and monoclonal CD3-PerCP-Vio700 antibody (clone BW264/56, Miltenyi). Cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer kit (eBioscience) and labeled using anti-IL-17A-APC-Vio770 (clone CZ8-2361, Miltenyi) and anti-TNF-α-eFlour 450 (clone Mab11, eBioscience) monoclonal antibodies. Cells were analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD). Data analysis was performed with FlowJo (LLC).
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2

Isolation and Cytokine Analysis of CD4+ T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Peripheral venous blood was obtained from study participants. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by density centrifugation using Biocoll (Merck, Darmstadt, Germany). 106 CD4+ enriched cell fractions isolated by CD4+ T Cell Kit (Miltenyi) were plated onto U-bottom plates and were restimulated for 4 hours at 37°C, 5% CO2 in a humidified incubator. Restimulation was conducted in a final volume of 200 μl RPMI 1640 (Sigma) supplemented with 10% FBS (Merck), 100 U/ml penicillin (Sigma), 100 mg/ml streptomycin (Sigma), 50 ng/ml PMA (Sigma), 250 ng/ml ionomycin (Sigma) and 1.3 μl/ml Golgistop (BD). After restimulation cells were stained with Life/Dead Fixable Aqua Dead cell kit (Thermo Fisher) and monoclonal CD3-PerCP-Vio700 antibody (clone BW264/56, Miltenyi). Cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer kit (eBioscience) and labeled using anti-IL-17A-APC-Vio770 (clone CZ8-2361, Miltenyi) and anti-TNF-α-eFlour 450 (clone Mab11, eBioscience) monoclonal antibodies. Cells were analyzed using a FACSCanto II flow cytometer and FACSDiva software (BD). Data analysis was performed with FlowJo (LLC).
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