Name: MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™
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Product data and images for MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™

Figures

Figure 1

Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant

^®

Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45

⁺

cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD

^–

cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter

^high

(SSC

^high

) cells, which correspond to low density PMN-MDSCs (region R5), CD14

⁺

cells (region R6) and SSC

^low

CD14

^–

cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14

⁺

HLA-DR

^–

. D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^–

CD33

^int

A) Gating strategy:
All events	Gated on R1
View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .	View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .
Gated on R2	Gated on R3
View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .	View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .
B) Identification of low density PMN cells (PMN-MDSCs):
Gated on R4	Gated on R5
View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .	View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .
C) Identification of M-MDSCs:
Gated on R6	Gated on R6
View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .	View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .
D) Identification of e-MDSCs:
Gated on R7	Gated on R7
View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .	View details MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™ Figure 1 Human PBMCs were stained using the MDSC Detection Kit, human and analyzed by flow cytometry using the MACSQuant ^® Analyzer 10. A) To exclude red blood cells and identify leukocytes, a first gate on CD45 ⁺ cells was set (region R1). Next, to eliminate doublets, a gate on single cells in Forward scatter-A (A=area) versus Forward scatter-H (H=height) was set (region R2). These cells were further distinguished from debris via Forward scatter-A and Side scatter-A (region R3). Afterwards, a gate on viable cells (7-AAD ^– cells) was set (region R4). B) Cells from region R4 were further separated into 3 subsets: side scatter ^high (SSC ^high ) cells, which correspond to low density PMN-MDSCs (region R5), CD14 ⁺ cells (region R6) and SSC ^low CD14 ^– cells (region R7). Low density PMN-MDSCs contained in region R5 were further separated into 4 subsets based on the expression of CD16 and CD11b (gates R8 to R11). C) Cells stained with MDSC Control Cocktail from R6 were displayed with CD14 versus REA Control-FITC, and a region enclosing >99.5% of cells was set (region R12). This region was transferred to the cells labeled with MDSC Staining Cocktail, depicting M-MDSCs as CD14 ⁺ HLA-DR ^– . D) Cells stained with MDSC Control Cocktail from region R7 were displayed with REA Control-FITC versus REA-Control-PE, and a region above the background staining, containing ^– CD33 ^int .

Specifications for MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™

Overview

The MDSC Detection Kit is developed to allow standardization and simplification of flow cytometric analysis of the subpopulations of MDSCs described so far. For greater flexibility, the product configuration also allows the extension of the analysis panel with additional specificities of interest (e.g. CD10, CD66b, CD15, and LOX-1).

Detailed product information

Background information

MDSC Phenotyping Cocktail, anti-human, REAfinity containing:

CD11b Antibody, anti-human, VioBlue®, REAfinity (clone: REA713, isotype: recombinant human IgG1)
CD45 Antibody, anti-human, VioGreen™, REAfinity (clone: REA747, isotype: recombinant human IgG1)
HLA-DR Antibody, anti-human, FITC, REAfinity (clone: REA805, isotype: recombinant human IgG1)
CD33 Antibody, anti-human, PE, REAfinity (clone: REA775, isotype: recombinant human IgG1)
CD16 Antibody, anti-human, PE-Vio® 770, REAfinity (clone: REA423, isotype: recombinant human IgG1)
CD14 Antibody, anti-human, APCVio 770, REAfinity (clone: REA599, isotype: recombinant human IgG1)
7-AAD Staining Solution

MDSC Control Cocktail, anti-human, REAfinity containing:

CD11b Antibody, anti-human, VioBlue, REAfinity (clone: REA713, isotype: recombinant human IgG1)
CD45 Antibody, anti-human, VioGreen, REAfinity (clone: REA747, isotype: recombinant human IgG1)
REA Control (S) Antibody, human IgG1, FITC, REAfinity (clone: REA293)
REA Control (S) Antibody, human IgG1, PE, REAfinity (clone: REA293)
CD16 Antibody, anti-human, PE-Vio 770, REAfinity (clone: REA423, isotype: recombinant human IgG1)
CD14 Antibody, anti-human, APCVio 770, REAfinity (clone: REA599, isotype: recombinant human IgG1)
7-AAD Staining Solution
CD16 Antibody, anti-human, PE-Vio 770, REAfinity (clone: REA423, isotype: recombinant human IgG1)
CD14 Antibody, anti-human, APC-Vio 770, REAfinity (clone: REA599, isotype: recombinant human IgG1)

Instrument details

Flow cytometer equipped with a red (640 nm), a blue (488 nm), and a violet (405 nm) laser.

Applications

Detection and quantification of MDSC subsets in human PBMCs. This product might be used for analysis of other starting materials, such as whole blood, lysed whole blood, or single-cell suspensions from human tissues. For such applications, optional reagents might be required, such as Red Blood Cell Lysis Solution (10×) or additional fluorochrome-conjugated antibodies.

Resources for MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™

Certificates

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References for MDSC Phenotyping Cocktail Kit, anti-human, REAfinity™

Publications

Marigo, I. et al. (2010) Tumor-induced tolerance and immune suppression depend on the C/EBPβ transcription factor. Immunity 32(6): 790-802
Marini, O. et al. (2017)
Mature CD10
⁺
and immature CD10
⁻
neutrophils present in G-CSF–treated donors display opposite effects on T cells.
Blood 129(10): 1343-1356
Veglia, F. et al. (2018) Myeloid-derived suppressor cells coming of age. Nat Immunol 19(2): 108-119
Mandruzzato, S. et al. (2016) Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study. Cancer Immunol. Immunother. 65(2): 161-169
Gabrilovich, D. I. et al. (2012) Coordinated regulation of myeloid cells by tumours. Nat. Rev. Immunol. 12(4): 253-268
Bruger, A. M. et al. (2020) Protocol to assess the suppression of T-cell proliferation by human MDSC. Meth. Enzymol. 632: 155-192
Lang, S. et al. (2018) Clinical relevance and suppressive capacity of human myeloid-derived suppressor cell subsets. Clin Cancer Res. 24(19): 4834-4844
Bronte, V. et al. (2016) Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun. 7(1): 12150
Condamine, T. et al. (2016) Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients. Sci Immunol 1(2): pii:aaf8943

Data and images