A Guide to Human Immune Cell Characterization by Flow Cytometry

Immune Cell Characterization by Flow CytometryFlow cytometry is a powerful technique that is widely used to identify and characterize different immune cell types in heterogeneous samples. It primarily relies on the use of fluorochrome-conjugated antibodies to detect the expression of specific cell surface or intracellular antigens on single cells in suspension. Using a combination of antibodies against different targets that are conjugated to fluorochromes with different emission wavelengths, multiple antigens can be detected simultaneously. As a result, distinct cell populations can be identified, quantified, and gated for further analysis.

Bio-Techne offers an unparalleled selection of fluorochrome-conjugated R&D Systems and Novus Biologicals antibodies qualified for flow cytometry that can be used to detect markers on different immune cell types present in mixed population samples, such as peripheral blood or tissue samples. Hundreds of world-renowned unique clones are available from the R&D Systems brand, many of which have been used to establish CD nomenclature through HLDA workshops. Additionally, the Novus Biologicals brand includes an expansive collection of both proprietary antibodies and some of the most highly referenced antibody clones on the market.

2

2

50K

0

100K

150K

200K

250K

SSC-

A

CD56

APC

CD19

AF5

94

CD8

PerC

P

CD25

Qdo

t 605

γδ T

CR

γδ T Cells

CD15+

Granulocytes

GranulocytesCD14+CD11b+ Monocytes

Tregs

Monocytes

Lymphocytes

CD15 AF488 CD14 AF405

CD4 AF750CD3 PE-Cy7

FSC-A

NK CellsB Cells

CD8+ T Cells

CD4+ T CellsT CellsCIKs

SSC-

A

CD11

b

AF7

00

10-Parameter Flow Cytometric Analysis of Immune Cells in Human Whole Blood. Human whole blood was stained with the following panel of monoclonal antibodies: Alexa Fluor®

700-conjugated Mouse Anti-Human CD11b/Integrin aM (R&D Systems, Catalog # FAB1699N), Alexa Fluor 405-conjugated Mouse Anti-Human CD14 (R&D Systems, Catalog # FAB3832V), Alexa Fluor 488-conjugated Mouse Anti-Human CD15/Lewis X (R&D Systems, Catalog # FAB7368G), PE-Cy7-conjugated Mouse Anti-Human CD3 (Novus Biologicals, Catalog # NB100-2726PECY7), Alexa Fluor 750-conjugated Mouse Anti-Human CD4 (R&D Systems, Catalog # FAB3791S), PerCP-conjugated Mouse Anti-Human CD8a (R&D Systems, Catalog # FAB1509C), Alexa Fluor 594-conjugated Mouse Anti-Human CD19 (R&D Systems, Catalog # FAB4867T), AmCyan-conjugated Anti-Human gdTCR, BV605-conjugated Anti-Human CD25/IL-2 Ra, and APC-conjugated Mouse Anti-Human CD56/NCAM-1 (R&D Systems, Catalog # FAB2408A). Different immune cell populations identified by the specific staining shown in each dot plot were gated and labeled or further stained as indicated to identify subsets of that particular cell type.

Identification of Human Granulocytic and Monocytic Myeloid-derived Suppressor Cells by Flow Cytometry. (A) Lin–/HLA-DR– cells were detected in human peripheral blood mononuclear cells by staining with a lineage cocktail containing Alexa Fluor 700-conjugated Mouse Anti-Human CD3e and CD19 Monoclonal Antibodies (R&D Systems, Catalog # FAB100N and # FAB4867N, respectively) and an Alexa Fluor 750-conjugated Mouse Anti-Human HLA-DR Monoclonal Antibody (R&D Systems, Catalog # FAB4869S). Lin–/HLA-DR– cells were gated. (B) CD11b+/CD33+ cells were detected in the Lin–/HLA-DR– cell population by staining with an APC-conjugated Mouse Anti-Human Siglec-3/CD33 Monoclonal Antibody (R&D Systems, Catalog # FAB1137A) and an Alexa Fluor 405-conjugated Mouse Anti-Human Integrin aM/CD11b Monoclonal Antibody (R&D Systems, Catalog # FAB16991V). CD11b+/CD33+ cells were gated. (C) Human granulocytic myeloid-derived suppressor cells (Lin–/CD11b+/CD14–/CD33+/CEACAM-8+/HLA-DR–) and monocytic myeloid-derived suppressor cells (Lin–/CD11b+/CD14+/CD33+/CEACAM-8–/HLA-DR–) were detected in the Lin–/HLA-DR–/CD11b+/CD33+ cell population by staining with a PE-conjugated Mouse Anti-Human CEACAM-8/CD66b Monoclonal Antibody (R&D Systems, Catalog # FAB4246P) and a PerCP-conjugated Mouse Anti-Human CD14 Monoclonal Antibody (R&D Systems, Catalog # FAB3832C).

Inte

grin

αM

/CD

11b

Siglec-3/CD33

CD14

CEACAM-8/CD66b

HLA

-DR

Lineage cocktail

Monocytic MDSCs

Granulocytic MDSCs

A B C

Cell Surface Markers Clone Fluorochrome-conjugated Antibodies

(Catalog # - Fluorochrome)

CCR2 48607* FAB150-R, S, T, U, V; FAB151-A, C, G, N, P

CCR3 61828* FAB155-A, C, F, G, P, R, S, T, U, V

CCR4 205410* FAB1567-A, C, F, G, N, P, R, S, T, U, V

CCR5 CTC5 FAB1802-A, F, G, N, P, R, S, T, U, V

CCR6 53103* FAB195-A, C, F, G, N, P, R, S, T, U, V

CCR7 150503* FAB197-A, F, G, N, P, R, S, T, U, V

CCR10 314305 FAB3478-A, G, N, P, R, S, T, U, V

CD1a 703217 FAB7076-A, G, N, P, R, S, T, U, V

CD3UCHT1* FAB100-A, C, F, G, N, P, R, S, T, U, V

OKT3 NBP2-24867-A, C, G, N, P, S, T, U, V **

CD434930 FAB379-G, N, R, S, T, U, V

RPA-T4 NBP2-27216-A, C, F, G, N, P, R, S, T, U, V **

CD8a

37006 FAB1509-A, C, F, G, N, P, R, S, T, U, V

53-6.7 NBP1-49045-A, C, G, N, P, R, S, T, U, V **

RPA-T8 NBP2-25195-A, C, F, G, N, P, R, S, T, U, V

CD11b/ Integrin aM

ICRF44 FAB1699-G, N, R, S, T, U, V

M1/70.15 NB600-1327-A, C, G, N, P, R, S, T, U, V **

CD11c ICRF3.9 FAB1777-A, C, G, N, P, R, S, T, U, V

CD14134620 FAB3832-A, C, F, G, N, P, R, S, T, U, V

M5E2 NB100-77758-A, C, F, G, N, R, S, T, U, V **

CD15 ICRF29-2 FAB7368-G, N, R, S, T, U, V

CD16/FcgRIII245536 FAB2546-A, C, F, G, N, P, R, S, T, U, V

3G8 NBP1-50056-A, C, F, G, N, P, R, S, T, U, V **

CD194G7-2E3 FAB4867-A, C, F, G, N, P, R, S, T, U, V

1D3 NBP2-24965-A, C, F, G, N, P, R, S, T, U, V **

CD20 396444 FAB4225-G, N, P, R, S, T, U, V

CD25/IL-2 Ra 24212 FAB1020-A, G, N, P, R, S, T, U, V

CD452D1 FAB1430-A, C, G, N, P, R, S, T, U, V

30-F11 NB100-77417-A, C, F, G, N, P, R, S, T, U, V **

CD45RA MEM-56 NB500-329-A, C, F, G, N, P, R, S, T, U, V **

CD45RO UCHL-1 NBP2-33104-A, C, F, G, N, P, R, S, T, U, V **

CD56/NCAM-1 301040 FAB2408-A, G, N, P, R, S, T, U, V

CD62L/L-Selectin 4G8 FAB9787-G, R, T

CD63460305 IC5048-G, N, P, R, S, T, U, V

H5C6 NBP2-42225-A, C, G, N, P, R, S, T, U, V **

CD66b/CEACAM-8 913542 FAB4246-A, G, N, P, R, S, T, U, V

CD68/SR-D1298807 IC20401-A, F, G, N, P, R, S, T, U, V

KP1 NB100-683-A, C, F, G, N, P, R, S, T, U, V **

Cell Surface Markers Clone Fluorochrome-conjugated Antibodies

(Catalog # - Fluorochrome)

CD69 298614 FAB23591-A, G, N, P, R, S, T, U, V

CD80/B7-1 37711 FAB140-F, G, N, P, R, S, T, U, V

CD86/B7-2 37301 FAB141-A, C, F, G, N, P, R, S, T, U, V

CD115/M-CSF R 61708 FAB329-A, F, G, N, P, R, S, T, U, V

CD117/c-kit47233 FAB332-A, G, N, P, R, S, T, U, V

2B8 NB100-77477-A, C, F, G, N, P, R, S, T, U, V **

CD123/IL-3 Ra 32703 FAB301-A, C, G, N, P, R, S, T, U, V

CD127/IL-7 Ra 40131 FAB306-A, G, N, P, R, S, T, U, V

CD141/BDCA-3 501733 FAB3947-A, G, N, R, S, T, U, V

CD163 215927 FAB1607-A, C, G, N, R, S, T, U, V

CD172/SIRPa 602411 FAB4546-G, N, P, R, S, T, U, V

CD206/MMR 685641 FAB25342-A, G, N, P, R, S, T, U, V

CLEC9a 683409 FAB6049-A, G, N, R, S, T, U, V

CRTH-2 301109 FAB33381-A, G, N, P, R, S, T, U, V

CXCR3 49801 FAB160--A, C, F, G, N, P, R, S, T, U, V

CX3CR1 528728 FAB5204-P

Fce RIa773704 FAB6678-C, G, N, R, S, T, U, V

AER-37 NBP1-43278-A, C, F, P **

HLA-DRL203 FAB4869-A, C, F, G, N, P, R, S, T, U, V

L243 NB100-77855-A, C, F, G, N, P, R, S, T, U, V **

Neuropilin-1/BDCA-4 446921 FAB3870-A, F, G, N, P, R, S, T, U, V

NKp44/NCR2 253415 FAB22491--A, G, N, P, R, S, T, U, V

XCR1 1097A FAB8571-G, N, P, R, S, T, U, V

Transcription Factors Clone Fluorochrome-conjugated Antibodies

(Catalog # - Fluorochrome)

AHR RPT9 NB300-515-A, G, N, P, R, S, T, U, V **

EOMES 644730 IC6166-A, G, N, R, S, T, U, V

FoxP31054C IC8214-A, G, N, P, S, T, U, V

376209R IC8970-G, N, R, S, T, U, V

GATA-3634919 IC63301-A, T

291106 IC26051-P

PU.1 732322 IC5870-G, N, R, S, T, U, V

RORgt1181A IC9125-A, G, N, R, S, T, U, V

600380 IC6006-A, C, P

T-bet

525803 IC5385-C, F, G, N, R, S, T, U, V

525831 IC53851-G, N, R, S, T, U, V

4B10 NBP1-43298-A, C, G, N, P, R, S, T, U, V **

Flow Cytometry Antibodies for Characterizing Human Immune Cell Types

Indicates a Novus Biologicals Antibody Indicates a R&D Systems Antibody* Clone was used by HLDA to establish CD designation. This antibody is either a recombinant monoclonal antibody or is available as a recombinant monoclonal antibody.Fluorochrome Key: A Allophycocyanin, C PerCP, F Fluorescein, G Alexa Fluor®488, N Alexa Fluor 700, P Phycoerythrin, R Alexa Fluor 647, S Alexa Fluor 750, T Alexa Fluor 594, U Alexa Fluor 350, V Alexa Fluor 405** Janelia Fluor and/or tandem dye conjugates are also available for these antibodies. Please visit novusbio.com for a complete listing of available fluorochromes.

Learn more| rndsystem.com/flowcytometry or novusbio.com/application/flow-cytometry

BR_Flow Guide_

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Flow Cytometry Handbook

Natural Killer Cell Markers

HematopoieticStem Cell

Common LymphoidProgenitor

Common MyeloidProgenitor

T Cells

Helper T Cells

Th1 Th2 Th9

Cytotoxic T Cells

B Cells NK Cells

Naive

Innate Lymphoid Cells (ILCs)

ILC1 ILC2

CD19+CD3+

CD56+CD8+

IFN-γ+

TCR-Vα24 Vβ11+

GATA-3+

IL-4+PU.1+

IL-9+

IL-4–

IL-17–

IFN-γ–

CD27–

IgD+

MemoryCD27+

CD23/Fcε RIIlow

CD138–

IgD–/+

CXCR3+

Tbet+

IFN-γ+

CD56–

CD117/c-kit–

CRTH-2–

NKp44–

EOMES–

CRTH-2+

CD117/c-kit+

GATA-3+

IL-4+

IL-5+

IL-13+

CCR6+

CD117/c-kit+

CRTH-2–

AHR+

RORγt+

NKp44+

IL-22+

NKp44–

IL-17+

PlasmaBCMA+

CD38+

CD138+

IgD–

RegulatoryCD24high

CD27+

IL-10+

Naive

CD45RA+, CD45RO–

CD62L+, CCR7+

CD45RO+, CD45RA–

CD62L–, CCR7–

CD45RA+, CD45RO–

CD62L–, CCR7–

CD45RO+, CD45RA–

CD62L+, CCR7+

Effector

Effector Memory (TEM)

Central Memory (TCM)

Type I (iNKT)

Type II NKT

ILC3

Highly Cytotoxic

(NCR+) ILC3

(NCR–) ILC3

CD3–

CD56+

CD127/IL-7 Rα–

Tbet+

EOMES+

(CD3/CD4/CD8/CD14/CD15/CD16CD19/CD33CD34/CD94/CD203c/Fcε RI)–

CD127/IL-7 Rα+

CD56low

CD16+

Weakly CytotoxicCD56high

CD16–

Th17

CCR4+

CCR6+

CCR10–

RORγt+

IL-17+

Th22

CCR10+

AHR+

IL-22+

IL-17–

IFN-γ–

TFH Treg

CXCR5+

ICOS+

PD-1+

Bcl-6+

IL-21+

CD25/IL-2 Rα+

CD127/IL-7 Rα–/low

FoxP3+

NKT Cells γδ T Cells

CD4+

T-bet+

IFN-γ+

TNF-α+

TCRγδ+

Mast Cells Basophils

Activated

Eosinophils Neutrophils Monocytes

Macrophages Monocyte-DerivedDendritic Cells

M1 Macrophage

CD1c/BDCA-1+

cDCs

M2 Macrophage

PlasmacytoidDendritic Cells

ClassicalDendritic Cells

PMN-MDSCs Mo-MDSCs

CD11b+ CD33/Siglec-3+, HLA-DR–

CD11b+ CD11b+

CD141/BDCA-3+

cDCs

Fcε RIα+

CD117/c-kithigh

CD63+

CD203c/ENPP-3+

Fcε RIα+

IL-3 Rα/CD123+

CD117/c-kit–

CCR3+

IL-5 Rα+

Siglec-8+

CD16/FcγRIII–

CD16/FcγRIII+

CD14–

CD15+

CD66b/CEACAM-8+

CD11c–

CD68+

HLA-DR+CD11c+

CD14+

CD1a+

CD1c/BDCA-1+

HLA-DR+

CD206+B7-1/CD80+

B7-2/CD86+

CD163–

iNOS+

CD163+

CD206+

CD14+

HLA-DR+

CD115/M-CSF R+

CD206– CD11clow

IL-3 Rα/CD123+

CLEC4C/CD303+

Neuropilin-1/BDCA-4+

CD11c+

HLA-DR+

CD1a–

CLEC9a–

CX3CR1+

SIRPα/CD172a+

XCR–

CD1a–

CLEC9a+

CX3CR1–

SIRPα/CD172a–

XCR1+

CD14–

CD15+

CD66b/CEACAM-8+

CD14+

CD15–

CD66b/CEACAM-8–

ClassicalCD14++

CD16/FcγRIII–

CCR2+

CX3CR1–

IntermediateCD14++

CD16/FcγRIII+

CCR2mid

CCR5+

CX3CR1high

Non-ClassicalCD14+

CD16/FcγRIII++

CCR2–

CX3CR1high

All leukocytes are CD45+

Cell Markers Guide for Human Immune Cell Characterization by Flow Cytometry

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