The Cytek^® 24-Color Mouse Immunoprofiling Panel is designed and optimized by Cytek scientists to enable researchers to identify more than 20 mouse immune subpopulations including T, B, NK cells, dendritic cells, monocytes, and granulocyte populations in fresh mouse samples. The Cytek^® 24-Color Mouse Immunoprofiling Panel has been optimized and titrated for analyzing fresh spleen, bone marrow, lymph node and blood samples on the Cytek Northern Lights^™ or Cytek Aurora^™ systems equipped with violet, blue, and red lasers.

The Cytek^® 24-Color Mouse Immunoprofiling Panel allows for the identification and characterization of multiple lineage subsets within mouse tissue suspensions including T, B, NK cells, dendritic cells, monocytes, and granulocyte populations in fresh mouse samples. The kit has been evaluated in fresh mouse splenocyte, bone marrow, lymph node and blood samples.

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PRODUCT DETAILS

Tested Dilution:  5 μL / test

Application: Flow Cytometry

Storage: 2-8°C and protected from Light. Do not freeze

Formulation: Phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide and 0.2% BSA (BSA Country of Origin USA)
*For Starbright^™ Dyes: Phosphate-buffered saline, pH 7.2, containing 0.1% PEG 3350, 0.1% Pluronic F-68, 0.05% Tween-20, 0.09% sodium azide and 1% BSA (BSA Country of Origin USA)

Data Sheets: TDS | SDS

DATA

Figure 1: Cell gating strategy for identifying subsets. Data were generated using fresh splenocytes from a C57BL/6 mouse, with red blood cells lysed. Sequential gates of red blood cell exclusion, singlets, and viable CD45⁺ cells are used to distinguish total live splenocytes. After excluding debris from the viable CD45⁺ events, NK cells and CD3⁺ NKT-like cells are identified by expression of CD49b vs. NK1.1, and subsequently gated on CD3 vs. B220. Basophils (CD49b⁺CD45^dim) are identified after excluding the 49b⁺NK1.1⁺ cells. Plasmacytoid dendritic cells (PDCs, CD317⁺⁺B220⁺) are determined after excluding the non-basophils. B cells are then identified in the non-PDC cells by positive expression of CD19 when gated vs. B220. From B220^-CD19^- gated events, γδT cells are identified by co-expression of CD3 and TCRγδ. From the non-γδ-T events, T cells are gated on the CD3 vs. TCR beta plot (gating on CD3⁺TCRb⁺). CD4⁺ and CD8⁺ T cell subsets are distinguished from the total T cell gate (CD4⁺CD8^- and CD8⁺CD4^- events) and then further subdivided into naïve and memory subsets by expression of CD62L and CD44 for each T cell subset (CD4⁺ and CD8⁺). CD4⁺CD25⁺ T cells (containing activated and regulatory T cells) can be identified from the CD4 vs. CD25 plot. Dendritic cells (DCs) are gated on a CD11c vs. I-A/I-E plot from the non-T cell events. DCs are further subdivided into cDC1 and cDC2 populations using CD8 vs. XCR1 and CD4 vs. CD11b plots respectively. From non-DC cells, neutrophils and eosinophils are identified using Siglec F (CD170) vs. Ly-6G. The Siglec F (CD170)/Ly-6G double negative events are then used to classify macrophages (MERTK⁺CD64^+/-) from the non-macrophage events. Total monocytes are identified by expression of CD11b⁺ and are subsequently gated to determine Ly-6C⁺ and Ly-6C^- monocyte subsets using CD11c and Ly-6C.

The panel contains the following single vial reagents.

Table 1: Cytek^® 24-Color Mouse Immunoprofiling Panel, cFluor^® Reagent Kit (22C) (P/N R7-40014), CD170 (Siglec F) Monoclonal Antibody (clone 1RNM44N), Brilliant Violet^™ 711, eBioscience^™ (P/N 407-1702-80), and MERTK Monoclonal Antibody (clone DS5MMER), Brilliant Violet^™ 480, eBioscience^™ (P/N 414-5751-80), and ViaDye^™ Red Fixable Viability Dye Kit (P/N R7-60008) are available to purchase separately.

RECOMMENDED USAGE

Fresh mouse splenocytes, bone marrow, lymph node and blood samples have been tested to optimize the performance of this kit. Please refer to the product web page for the staining protocols, fluorochrome list, experiment template and data acquisition protocol.

Please briefly centrifuge the reagent vial before use.

Use appropriate personal protective equipment per the product safety data sheet when using this product.

• Download the Sample Preparation Guidelines for the Cytek^® 24-Color Mouse Immunoprofiling Panel
  
• Download the Acquisition Protocol for the Cytek^® 24-Color Mouse Immunoprofiling Panel
• Download the Cytek^® 24-Color Mouse Immunoprofiling Panel Experiment Template for:
  • Splenocytes
  • Lymph Node
  • Blood
  • Bone Marrow
• Download the Cytek^® 24-Color Mouse Immunoprofiling Panel Assay Tags

REFERENCES
Hornsteiner, F, et al. Eur J Immunol. 52, 2006 (2022)
Vanekova, L, et al. Methods Protoc. 5, 70 (2022)
Becht, E, et al. Sci Adv. 7, 1 (2021)
Origin A, et al. Nat Rev Immunol. 3, 583 (2003)
Yu Y-RA, et al. PLOS One. 11 (3), (2016)
Gautier EL, et al. Nat Immunol. 13, 1118 (2012)
Asselin-Paturel C, et al. Immunol. 171, 6466 (2003)
Dong, MB, et al. J Immunol Methods. 432, 4 (2016)45
Yang, S-Y, et al. J Immunol Methods. 507, 4 (2022)
Brandi, J, et al. Cytometry Part A. May (2023)
Mestas, J, et al. J Immunol. 172, 2731 (2004)
Ardavin, C, et al. Nat Rev Immunol. 3, 582 (2003)
Wu, Y-C, et al. J Immunol Methods. 515, 1 (2023)

For Research Use Only. Not intended for use in diagnostic procedures.

¹cFluor^® V547, cFluor^® B515, cFluor^® B532, cFluor^® B548, cFluor^® BYG610, cFluor^® BYG645, cFluor^® R685, cFluor^® R720 and cFluor^® R840 are equivalent to CF^®405L, CF^®488A, CF^®503R, CF^®514, RPE-CF^®596R, cFluor^® BYG610, CF^®660C, CF^®700 and APC-CF^®790T, respectively, manufactured and provided by Biotium, Inc. under an Agreement between Biotium and Cytek (LICENSEE). The manufacture, use, sale, offer for sale, or import of the product is covered by one or more of the patents or pending applications owned or licensed by Biotium. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim, no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.

Cytek^® cFluor^® V435, cFluor^® V605, cFluor^® V670, cFluor^® V780 are StarBright^™ Violet 440, StarBright^™ Violet 610, StarBright^™ Violet 670, StarBright^™ Violet 790, respectively, manufactured and provided by Bio-Rad, Inc.

StarBright is a trademark of Bio-Rad Laboratories, Inc. in certain jurisdictions. StarBright Dyes are manufactured and provided by Bio-Rad under an Agreement between Bio-Rad supplied to a company, being subject to certain conditions.

²cFluor^® BYG610, cFluor^® BYG645, cFluor^® BYG667, cFluor^® BYG710, cFluor^® BYG750, and cFluor^® BYG781 are tandem dyes made with R-PE. cFluor^® R780 and cFluor^® R840 are tandem dyes made with APC. Caution – Tandem dyes may show changes in their emission spectra with prolonged exposure to light or fixatives.