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Dendritic Cell Phenotyping Kits


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Imgenex introduced new antibody mixes and kits for flow cytometry cell phenotyping.

LMAX Human Lineage Marker Antibody Mix


(IMG-6628k 25 tests)
The human Lineage Marker Antibody Mix contains 25 tests of each antibody, tested and standardized to use at 10 µl, to accommodate multi-color assay formats. Human LMAX (hLMAX) contains FITC-conjugated antibodies that, when added with HLA-DR as an additional marker, can be used to identify, enrich and/or deplete T, B, NK, and precursor myeloid cells (such as monocytes). The reagent can be used at 10 µl per one million cells in 100 µl total staining volume (or 100 µl whole blood). IMGENEXs red blood cell lysis buffer (Cat # 10089) is recommended for lysing RBCs. Human FITC Lineage Markers and PerCP-Cy5.5 HLA-DR monoclonal antibody mix has been validated in flow cytometric analysis of Plasmacytoid Dendritic Cells from: whole peripheral blood within 24h of collection, fresh collected PBMCs, frozen PBMCs. This kit can also be used for the analysis of Myeloid Dendritic Cells.

Plasmacytoid Dendritic Cell (pDC) Identification Kit


(IMG-6629k 25 tests)
Contains 25 tests of hLMAX (FITC Lineage Marker & PerCP-Cy5.5 HLA-DR monoclonal antibody mix) plus CD123 AF647 plus IgG1 AF647 (isotype control). Standardized to use at 10 µl, to accommodate multi-color assay formats. The contents of this kits can be used to identify plasmacytoid Dendritic Cells (pDCs) from human blood. The human Plasmacytoid DC Identification Kit has been validated in flow cytometric analysis of: whole peripheral blood within 24h of collection, fresh collected PBMCs, frozen PBMCs. Plasmacytoid Dendritic Cells (pDCs) are a subset of peripheral blood Dendritic Cells which play an important role in both innate and adaptive immune responses. pDCs recognize viral components through Toll-like Receptors such as TLR7, TLR9 and others and rapidly produce Interferon-alpha as a primary anti-viral immune response. pDCs also participate in antigen presentation, an important functional role which drives the adaptive immune response. Intense investigation has led to pDC identification by a unique set of markers by flow cytometry. pDCs are usually present in relatively low frequency in peripheral blood. Direct identification of subsets of peripheral blood DCs has an advantage in analyzing changes in number or function of DC subsets during chronic viral infections or diseased patients, as examples.

Plasmacytoid Dendritic Cell (pDC)/Toll-like Receptor 9 Investigation Kit


(IMG-6630k 25 tests)
Contains 25 tests of hLMAX (FITC Lineage Marker & PerCP-Cy5.5 HLA-DR monoclonal antibody mix), CD123 AF647, IgG1 AF647 (isotype control), TLR9 PE, IgG1 PE (isotype control), Staining buffer, Fixation buffer, Permeabilization buffer. The contents of this kits can be used to identify TLR9 in plasmacytoid Dendritic Cells (pDCs) from human blood. The human Plasmacytoid DC/TLR9 Kit has been validated in flow cytometric analysis: whole peripheral blood within 24h of collection, fresh collected PBMCs, frozen PBMCs. Plasmacytoid Dendritic Cells (pDCs) are a subset of peripheral blood Dendritic Cells which play an important role in both innate and adaptive immune responses. pDCs recognize viral components through Toll-like Receptors such as TLR7, TLR9 and others and rapidly produce Interferon-alpha as a primary anti-viral immune response. pDCs also participate in antigen presentation, an important functional role which drives the adaptive immune response. Intense investigation has led to pDC identification by a unique set of markers by flow cytometry. pDCs are usually present in relatively low frequency in peripheral blood. Direct identification of subsets of peripheral blood DCs has an advantage in analyzing changes in number or function of DC subsets during chronic viral infections or diseased patients, as examples.

CD123 TLR9 Phenotyping
Human PBMCs were surface stained with hLMAX FITC Lineage & PerCP-Cy5.5 HLA-DR monoclonal antibody mix), CD123 AF647 (or isotype IgG1 AF647) by following a standard staining protocol. The dot plots shown demonstrate the identification of pDC (CD123+ cells). TLR9 expression by pDC is shown using TLR9 PE (Cat # IMG-305D) along with Isotype control PE.

The human Plasmacytoid DC/TLR9 Kit has been validated in flow cytometric analysis:
  • whole peripheral blood within 24h of collection
  • fresh collected PBMCs
  • frozen PBMCs
IMGENEX’s red blood cell lysis buffer (Cat # 10089) is recommended for lysing RBCs. IMGENEX’s cell surface flow cytometry kit (Cat # 10084K) was used for the experiment shown above.

Background


Plasmacytoid Dendritic Cells (pDCs) are a subset of peripheral blood Dendritic Cells which play an important role in both innate and adaptive immune responses. pDCs recognize viral components through Toll-like Receptors such as TLR7, TLR9 and others and rapidly produce Interferon-alpha (IFNα) as a primary anti-viral immune response. pDCs also participate in antigen presentation, an important functional role which drives the adaptive immune response. Intense investigation has led to pDC identification by a unique set of markers by flow cytometry. pDCs are usually present in relatively low frequency in peripheral blood. Direct identification of subsets of peripheral blood DCs has an advantage in analyzing changes in number or function of DC subsets during chronic viral infections or diseased patients, as examples.

Testing human pDC/TLR9 kit using whole blood and freshly isolated PBMCs


Protocol:
  1. Label tubes as 1, 2, 3 & 4 (use regular FACS tubes 12x75)
  2. Dispense 100 μl of whole blood or 1 x 10^6 PBMCs into each tube
  3. Add hLMAX mix (10 μl) into 2, 3, 4 and mix (vortex) gently
  4. Add IgG1 AF647 (1 μg) into Tube 2 and mix (vortex) gently
  5. Add CD123 AF647 (1 μg) into Tubes 3 & 4, mix (vortex) gently
  6. Mix tubes gently and leave them protected from light in dark (cover with foil) at room temperature for 20 min (if using PBMCs, proceed to step 10)
  7. Add 2 ml of 1X RBC lysis buffer to each tube, gently vortex, and incubate at RT for another 10 min (dark, cover with foil)
  8. Centrifuge at 1000 RPM for 10 min
  9. Gently aspirate the supernatant, leaving as little as possible (100-200 μl)
  10. Add 2 ml of 1X FACS staining buffer to each tube and centrifuge at 1200 RPM for 10 min
  11. Repeat the step 10
  12. Suspend the pellet in tube 1, 2, 3 in 300 μl of 1X staining buffer
  13. Suspend the pellet in tube 4 in 300 ul of 1X fixation buffer, gently vortex and leave them on ice for 20 min. Vortex samples once again after 10 min and continue incubation (for a total of 30 min)
  14. Add 1.5 ml of 1X permeabilization buffer and centrifuge at 1200 RPM for 10 min
  15. Gently decant the sup and add 1 ml of perm buffer and incubate on ice for 10 min
  16. Wash with 1X perm buffer (2x)
  17. Divide the pellet into 3 parts of 100 ul each, label as 4a, 4b & 4c
  18. Add 20 ul of IgG1 PE (1 μg diluted into 100 ul perm buffer) and vortex
  19. Add 20 ul of TLR9 PE (1 μg diluted into 100 ul perm buffer) and vortex
  20. Incubate on ice for 30 min
  21. Wash twice with 1X perm buffer
  22. Suspend in 300 μl of 1X staining buffer
  23. Analyze samples along with single color stained tubes, prepared in similar way
  24. Prepare separate single color tubes for surface and IC staining, as they differ in FSC/SSC.

G4 Dendritic Cell Generation Kit

 



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