Upstream / Downstream

pathwayImage

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

Questions?

Find answers on our FAQs page.

ANSWERS  

Visit PhosphoSitePlus®

PTM information and tools available.

LEARN MORE

Figure 1. Target map of the PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) #13124.

Learn more about how we get our images

Figure 2. Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stimulated with immobilized anti-human CD3 antibody, soluble anti-human CD28 antibody (1 μg/ml), hIL-2 #8907 (2 ng/ml) and hIL-4 #8919 (1 ng/ml) for 2 days. Cells were washed and cultured for 3 days with hIL-2 (2 ng/ml) and hIL-4 (1 ng/ml). Cells were then washed and stimulated with TPA #4174 (10 ng/ml) and Ionomycin, Calcium Salt #9995 (1 μg/ml) for 24 hr before supernatant collection. Supernatants were prepared and analyzed using PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) #13124. Panel A shows images that were acquired using LI-COR® Biosciences Odyssey® imaging system. Panels B and C show high and low end raw values of quantified fluorescence intensity. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.

Learn more about how we get our images

Figure 3. Human PBMCs were isolated from whole blood and pre-treated with hIFN-γ #8901 (10 ng/ml) for 2 hr. LPS was then added (1 μg/ml) and incubated for an additional 22 hr before supernatant collection. Supernatants were prepared and analyzed using the PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) #13124. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows raw values of quantified fluorescence intensity. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.

Learn more about how we get our images

Figure 4. Human PBMCs were isolated from whole blood and CD4+ T cells separated by positive selection. The CD4+ T cells were stimulated with immobilized anti-human CD3 antibody, soluble anti-human CD28 antibody (1 μg/ml), hIL-23 (10 ng/ml), hTGF-β1 #8915 (1 ng/ml), hIL-6 #8904 (20 ng/ml), hTNF-α #8902 (10 ng/ml), hIL-1β #8900 (10 ng/ml), anti-human IL-4 antibody (10 μg/ml) and anti-human IFN-γ antibody (1 μg/ml) for 3 days before supernatant collection. Supernatants were prepared and analyzed using PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) #13124. Panel A shows images that were acquired using LI-COR® Biosciences Odyssey® imaging system. Panels B and C show high and low end raw values of quantified fluorescence intensity. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.

Learn more about how we get our images
Image
Page

PathScan® Th1/Th2/Th17 Cytokine Antibody Array (Fluorescent Readout) #13124 Protocol

A. Preparing Cell Culture Supernatants

  1. Culture cells as desired.
  2. Collect the conditioned media and centrifuge to clarify. Transfer the clarified conditioned media to a new tube. It may be used immediately or stored at -80°C in single-use aliquots. Avoid multiple freezing and thawing.
  3. Immediately before performing the assay, dilute the clarified conditioned media accordingly in Array Diluent Buffer. Undiluted clarified conditioned media can also be used.
  4. Set aside on ice.

B. Assay Procedure

  1. Bring glass slides and blocking buffer to room temperature before use.
  2. Prepare 1X Array Wash Buffer by diluting 20X Array Wash Buffer in deionized water. Dilute 2.5 ml of 20X Array Wash Buffer with 47.5 ml of deionized water. Label as 1X Array Wash Buffer and keep at room temperature.
  3. Prepare 1X Detection Antibody Cocktail as follows:
    • For running only 1 slide: Dilute 150 μl of 10X Detection Antibody Cocktail with 1350 μl of Array Diluent Buffer. Keep 1X Detection Antibody Cocktail on ice.
    • For running 2 slides: Dilute 300 μl of 10X Detection Antibody Cocktail with 2700 μl of Array Diluent Buffer. Keep 1X Detection Antibody Cocktail on ice.
  4. Prepare 1X DyLight™ 680-linked Streptavidin as follows:
    • For running only 1 slides: Dilute 150 μl of 10X DyLight™ 680-linked Streptavidin with 1350 μl of Array Diluent Buffer. Keep 1X DyLight™ 680-linked Streptavidin on ice and protect from light.
    • For running 2 slides: Dilute 300 μl of 10X DyLight™ 680-linked Streptavidin with 2700 μl of Array Diluent Buffer. Keep 1X DyLight™ 680-linked Streptavidin on ice and protect from light.
  5. Affix the multi-well gasket to the glass slide (see figure at right):
    1. Place the multi-well gasket face-down on the bench top, with the silicone layer should be facing up. Remove the protective plastic film.
    2. Carefully place the glass slide on top of the multi-well gasket with the nitrocellulose pads facing down while aligning the pads with the openings in the gasket. The orientation line should appear in the upper left hand corner when the slide is oriented vertically.
    3. Insert the metal clip into the groove in the gasket and rotate the clip into the locked position. Ensure that the clip is on the same side as the orientation line on the slide.

      Note: one of the clips has a small dot etched onto the upper rib to assist with pad designation (see slide assembly photos).

    4. Slide the clip into place.
    5. Snap the second metal clip to the other side of the assembly in the same manner and slide into place.
    6. The assembled array is ready to use.
  6. Add 100 μl Array Blocking Buffer to each well and cover with sealing tape. Incubate for 15 min at room temperature on an orbital shaker.

    Note: Do not allow the pads to dry out at any time during the assay.

  7. Decant Array Blocking Buffer by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 75 μl of clarified conditioned media to each well and cover with sealing tape. Incubate for 2 hr at room temp (or overnight at 4°C) on an orbital shaker.
  8. Decant well contents by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 100 μl 1X Array Wash Buffer to each well and incubate for 5 min at room temperature on an orbital shaker. Repeat three more times. Decant well contents.
  9. Add 75 μl 1X Detection Antibody Cocktail to each well and cover with sealing tape. Incubate for 1 hr at room temperature on an orbital shaker.
  10. Wash 4 times for 5 min each with 100 μl 1X Array Wash Buffer as in step 8.

    Note: From this point on, keep slide protected from light.

  11. Add 75 μl 1X DyLight™ 680-linked Streptavidin to each well and cover with sealing tape. Incubate for 30 min at room temperature on an orbital shaker.
  12. Wash 4 times for 5 min each with 100 μl 1X Array Wash Buffer as in step 8.
  13. Remove multi-well gasket by pulling the bottom of the metal clips away from the center of the slide, then peeling the slide and gasket apart.
  14. Place the slide face up in a plastic dish (a clean pipette tip box cover works well). Wash once for 10 sec with 10 ml deionized water.
  15. Remove slide from plastic dish and allow to dry completely.
  16. Capture an image of the slide using a fluorescent digital imaging system capable of exciting at 680 nm and detecting at 700 nm. Quantify spot intensities using commercially available array image analysis software.
Slide Assembly Photos

DyLight 680™ is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.

Product Includes Quantity Cap Color
16-Well Gasket 2 Ea
Sealing Tape 2 sheets
20X Array Wash Buffer 15 ml White
Array Blocking Buffer 5 ml Red
Array Diluent Buffer 15 ml Blue
DyLight 680TM-linked Streptavidin (10X) 300 µl Brown

Product Usage Information

Storage: Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).

Product Description

Description: The PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. This array kit allows for the simultaneous detection of 12 unique extracellular signaling molecules. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two slides allowing the user to test up to 32 different samples and generate 384 data points in a single experiment. Cell supernatant is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated Dylight™ 680 is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.


Specificity / Sensitivity

PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit (Fluorescent Readout) detects the target proteins as specified on the Array Target Map (figure 1). No significant cross reactivity has been observed between targets. This kit is optimized for cell culture supernatants. Recommended starting cell culture supernatant range is 20-75 μl. All antibodies have been validated for human-derived cell culture supernatants.


Species Reactivity: Human

Cytokines are secreted intercellular signaling molecules that regulate many biological processes including inflammation, host defense, and cell differentiation. Cytokine profiles may provide insight into the molecular mechanisms that distinguish between healthy and diseased states. The PathScan® Th1/Th2/Th17 Cytokine Antibody Array Kit offers an antibody panel against a broad array of cytokines to enable measurement of their relative changes in cell culture supernatants.

Upon activation, naive CD4+ helper T cells differentiate into distinct functional subsets. The development of these subsets is driven, in part, by the cytokine milieu. Type 1 (Th1) cells help drive cellular immunity against intracellular pathogens. IL-12 and IFN-γ induce Th1 cell development. Th1 cells produce IFN-γ and IL-2, which provide a positive feedback loop to enhance Th1 cell differentiation and NK cell and CD8+ T cell cytolytic activity.

Th2 cells play a crucial role in the humoral immune response against extracellular pathogens. IL-4 drives development of Th2 cells, which subsequently produce IL-4, IL-5, and IL-13. These cytokines induce B cell proliferation, antibody production, IgE class switching, and activate eosinophils respectively.

Another distinct helper T cell lineage, Th17, is important for mucosal immunity. Dysregulation of Th17 may significantly contribute to the development of autoimmunity. IL-17 produced by Th17 cells induce secretion of pro-inflammatory cytokines IL-6, IL-8, GM-CSF, and TNF-α. Many of these molecules link innate and adaptive immunity through the recruitment and activation of innate immune cells.

Effective immune responses require finely tuned coordination between pro and anti-inflammatory signals. Pro-inflammatory molecules play important roles in activating key immune players to fight infection. IL-8 induces granulocyte migration and activates neutrophil phagocytic activity. GM-CSF mobilizes monocytes into infected tissue and activates macrophage and neutrophils. TNF-α is a multifunctional pro-inflammatory cytokine involved with a number of processes including cell proliferation, differentiation, and apoptosis.

Uncontrolled inflammation may damage surrounding host tissue. IL-10 is a prototypical anti-inflammatory cytokine that serves to terminate the acute inflammatory response by inhibiting Th1 cell function and pro-inflammatory cytokine production.


1.  Romagnani, S. (1999) Inflamm Bowel Dis 5, 285-94.

2.  Bradley, L.M. et al. (2000) Immunol Res 21, 149-58.

3.  O'Garra, A. and Arai, N. (2000) Trends Cell Biol 10, 542-50.

4.  Harrington, L.E. et al. (2005) Nat Immunol 6, 1123-32.

5.  Stockinger, B. and Veldhoen, M. (2007) Curr Opin Immunol 19, 281-6.

6.  Köhidai, L. and Csaba, G. (1998) Cytokine 10, 481-6.

7.  Carey, A.J. et al. (2012) JAKSTAT 1, 159-167.


Entrez-Gene Id 1437 , 3458 , 3586 , 3592 , 3596 , 3605 , 3558 , 3565 , 3567 , 3569 , 3576 , 7124
Swiss-Prot Acc. P04141 , P01579 , P22301 , P29459 , P35225 , Q16552 , P60568 , P05112 , P05113 , P05231 , P10145 , P01375


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
LI-COR is a registered trademark of LI-COR, Inc.
Odyssey is a registered trademark of LI-COR, Inc.