mouse ifnγ il4 double color elispot plates  (Cellular Technology Ltd)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 1. Coexpression of type 1 and type 2 cytokines in Th1 and Th2 clone as studied by intracytoplasmic staining and two-color ELISPOT analysis. Th1 clone SH10 (A and B) and Th2 clone M33 (C and D) were cultured with unirradiated, T cell-depleted, syngeneic LN cells from wild- type mice with and without Ag, and the induction of cytokine production was measured in parallel by intracytoplasmic FACS analysis or two-color ELISPOT analysis detecting IL-2 and IFN-g (A and B) and IL-4 and IL-5 (C and D). For intracytoplasmic staining, 3 3 105 T cells/ml were cultured with 1 3 106 APC/ml with and without Ag: results are shown for the T cell-gated population. Results are shown for the stimulated cultures with the gates set using the nonstimulated cultures with relevant Abs and isotype controls. For the ELISPOT assays, the indicated numbers of T cells were plated per well with the numbers of APC constant at 1 3 106 cells per well. The SD shown is from triplicate wells. The least square approximation of the first order was used (Y 5 aX 1 b) to approximate these data. The correlation coefficients for all graphs shown (r) were .0.995 and b 5 0 6 2, showing that the graphs were linear and passed through the origin. The results are from one experiment that is representative of four performed. An example of two-color image analysis for B is provided in Fig. 2.

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Staining, Enzyme-linked Immunospot, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 2. Computerized image analysis of two- color ELISPOT assay. Enlarged image fragments of IFN-g/IL-2 assay wells from Fig. 1B are shown to il- lustrate single-positive spots and several shades of dou- ble-positive spots (IFN-g in red, IL-2 in blue). Unproc- essed images of medium-control well (A) and of Ag- stimulated well (B) both containing 125 SH10 T cells and 106 APC/well are shown. Consecutive steps of im- age analysis are shown (C–F) with the image analyzer detecting red, blue, and double-colored spots separately by three different threshold settings. C, With the blue threshold active, single-color blue and all double-posi- tive spots (which have blue as a part of their color com- position) are detected and outlined, for better visibility highlighted with arrows. Similarly, under the red thresh- old (D), only red single-color and double-color spots are detected. E, The double-color threshold shown is a mathematical intersection of the two single-color thresh- olds. F, The final step of analysis, with single-color and double-color spots highlighted with artificial colors: blue, red, and green, for single-blue, single-red, and double-color spots, respectively. Counted spots are la- beled with letter symbols of the corresponding color. To eliminate the assessment of partially overlapping red and blue spots as double-color spots, the image analyzer counts only double-color spots that are formed by the color mixture of concentric blue and red spots with sin- gle dense centers. Further detail on image analysis is provided in Materials and Methods.

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Enzyme-linked Immunospot, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 3. Spot size distribution of cloned T cells and freshly isolated CD4 cells representing the spectrum of cytokine output per cell. A, Ag- induced IFN-g spots produced by cloned SH10 T cells (F) and by purified CD4 LN cells isolated from OVA:CFA-immunized BALB/c mice (M) were categorized according to size. The size histograms generated by the analyzer represent the distribution of spots falling into 15 size categories (the symbols) between 1023 and 1 mm2. Spots of area less than 1022 mm2

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Clone Assay, Isolation, Produced, Generated

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 4. Polarized and uninhibited production of IL-5 or IFN-g in freshly isolated spleen cells. BALB/c mice were immunized with either OVA:IFA (A) or OVA:CFA (C) and their spleen cells were tested on day 21 in cultures containing either medium alone (the wells on the left) or OVA (all other wells) in a two-color cytokine ELISPOT assay: IL-5 in red, IFN-g in blue. The cells from the primed mice were plated either undiluted, at 1 3 106 cells/well, or in two-fold serial dilutions with unirradiated naive BALB/c spleen cells, thus keeping the total cell number constant at 1 3 106 cells/well. The cell dilutions in A progress from the left to the right and, in C, from the right to the left. The number (3105) of cells derived from mice primed with either OVA:IFA or OVA:CFA is shown, respectively, in the upper right or lower left corner of each panel showing a representative well. B, The spleen cells from the mice primed with OVA:IFA and OVA:CFA were mixed such that the cell numbers correspond to the numbers shown in the panel above (A) and below (C) the image. Beneath each panel, in the respective colors, the mean 6 SD of red or blue spots in quadruplicate wells is shown. The data are representative of two experiments performed with unseparated spleen cells and one each with purified CD4 cells and Th1/Th2 clones.

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Isolation, Enzyme-linked Immunospot, Derivative Assay, Clone Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 6. Dose-response characteristic of the type 1 cytokine response of memory cells primed by different immunization doses. Groups of four BALB/c mice were immunized with doses of 0.1–100 mg of OVA323–339 in CFA, as indicated, and LN cells (1 3 106/well) were tested for the recall response 21 days later. A, The production of the cytokine indicated was tested at a 10-mM recall dose of the peptide. The data are from individual mice representative of three independent experiments. B–D, Dose response for the indicated cytokines and cytokine pairs in mice immunized as specified in the inserts. Because cells producing IL-4 and IL-5 were not detected in significant numbers at any recall concentration, these data are not shown. SD for triplicate wells were in the range of 5–7%. For each immunizing dose, the cytokine recall response of a single representative mouse is shown. The data were re- produced in three experiments performed on day 21.

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Concentration Assay, Produced

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Single-cytokine-producing CD4 memory cells predominate in type 1 and type 2 immunity.

doi: 10.4049/jimmunol.164.4.1862

Figure Lengend Snippet: FIGURE 8. Overall frequency of CD4 cells coexpressing select cyto- kine pairs. The cumulative data from multiple experiments are shown for CD4 memory cells induced by immunization with OVA protein or OVA323–339 in IFA or CFA and those induced in the course of Leishmania infection. The percentage of coexpressing cells from the total number of cytokine-producing CD4 cells is shown, as specified in Table I. The mean values and SD were calculated from the data averaged from the individual experiments for each of these models and represent between 20,000 and 600,000 spots, depending on the individual cytokine combinations.

Article Snippet: Double-color cytokine ELISPOT assays Plates (ImmunoSpot M200; Cellular Technology Limited, Cleveland, OH) were coated overnight at 4°C with the cytokine-specific capture Abs specified below.

Techniques: Infection