BD PharmingenTM PE Rat Anti-Mouse IL-10

Description

Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest. Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest. Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest. Show More Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest. Show More Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest. Expression of IL-10 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 clone 145-2C11, Cat. No. 553058 at 25 mug/ml and soluble anti-mouse CD28 clone 37.51, Cat. No. 553294 at 2 mug/ml for 2 days in culture together with recombinant mouse IL-2 10 ng/ml, Cat. No. 550069 and recombinant mouse IL-4 1 ng/ml, Cat. No. 550067 , followed by a 3 day incubation with only IL-2 and IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 25 mug/ml and anti-mouse CD28 2 mug/ml in the presence of GolgiStop Cat. No. 554724 . The cells were then stained with 0.06 mug of FITC-conjugated rat anti-mouse CD4 FITC-RM4-5, Cat. No. 553046 and 0.12 mug of PE-conjugated rat anti-mouse IL-10 antibody PE-JES5-16E3, Cat. No. 554467 by using BD Pharmingens staining protocol. To demonstrate specificity of staining, the binding of PE-JES5-16E3 was blocked by the preincubation of the conjugated antibody with molar excess of recombinant mouse IL-10 0.12 mug, Cat. No.550070; middle panel , and by preincubation of the fixed and permeabilized cells with an excess of the unlabelled JES5-16E3 mAb 3.6 mug, Cat. No. 554464; right panel . The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking middle panel and unlabelled antibody blocking right panel specificity controls. A suitable rat IgG2b isotype control for assessing levels of background staining on fixed/permeabilized mouse cells is PE-R35-38 Cat. No. 556925 ; use at comparable concentrations to antibody of interest.