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Immunophenotyping
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Immunophenotyping

Immunophenotyping by flow is the process of using a cells surface markers to classify individual cells from a population into more delineated subgroups. For example, taking white bloods cells from a sample and subgrouping them into B cells or T cells. You can then further group them into CD4 or CD8 T cells, and further then into Effector CD4 T cells, Memory CD4 T cells and so on. This process uses fluorescently labeled antibodies specific for these cell surface markers, and then “gating” on these fluorescent antibodies in ways to discriminate the specific populations of interest. More complex sample types, or the more specific a population of interest is, the better the immunophenotyping antibody panel needs to be. And that is where RayBiotech comes in. Our service team has a number of preset and validated panels for your population, and if we don’t, we can make one tailored to your project needs. We have validated panels as well for human, mice, and rats, but can also consider other model systems as the cases arise. See some examples below, or reach out and let us know what panel you’d like to use.

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Immunophenotyping Panels

The major immune cell lineages and certain subsets can be identified by this panel, which only involves cell surface marker staining. Covers the populations listed below​

Human

Cell Type

Major Markers

Lineage

T cell

CD45+ CD3+ CD4+ CD8+

Lymphoid

B cell

CD45+ CD19+

NK cell

CD45+ CD3- CD19- CD56+ CD16+/-

Monocyte

CD45+ CD3- CD19- CD56- CD14+ CD16+/-

Myeloid

Macrophage

CD45+ CD3- CD19- CD56- CD11b+ CD68+

DC

CD45+ CD3- CD19- CD56- CD11c+ HLA-DR+

Mast cell

CD45+ CD3- CD19- CD56- CD11b- CD117+

Basophil

CD45+ CD3- CD19- CD56- CD11b- FCεRIα+

Eosinophil

CD45+ CD3- CD19- CD56- CD11b+ CD49d+ CD16-

Neutrophil

CD45+ CD3- CD19- CD56- CD11b+ CD49d- CD16+

Mouse

Cell Type

Major Markers

Lineage

T cell

CD45+ CD3+ CD4+ CD8+

Lymphoid

B cell

CD45+ CD45R+

NK cell

CD45+ CD3- CD45R- CD49b+

Monocyte

CD45+ CD3- CD45R- Ly6C+ CD11b+

Myeloid

Macrophage

CD45+ CD3- CD45R- CD11b+ CD68+

DC

CD45+ CD3- CD11c+ CD11b+/-

Mast cell

CD45+ CD3- CD45R- CD11b- CD117+

Basophil

CD45+ CD3- CD45R- CD11b- FCεRIα+

Eosinophil

CD45+ CD3- CD45R- CD11b+ F4/80+ Ly6G-

Neutrophil

CD45+ CD3- CD45R- CD11b+ F4/80- Ly6G+

Investigator can choose different markers in this panel (lineage markers are in gray) to identify major immune cell lineages. Up to 20 colors including viability dye can be tested at one time!

The major immune cell lineages and certain subsets can be identified by this panel, which only involves cell surface marker staining. Covers the populations listed below​

Human

Cell Type

Major Markers

Lineage

T cell

CD45+ CD3+ CD4+ CD8+

Lymphoid

B cell

CD45+ CD19+

NK cell

CD45+ CD3- CD19- CD56+ CD16+/-

Monocyte

CD45+ CD3- CD19- CD56- CD14+ CD16+/-

Myeloid

Macrophage

CD45+ CD3- CD19- CD56- CD11b+ CD68+

DC

CD45+ CD3- CD19- CD56- CD11c+ HLA-DR+

Mast cell

CD45+ CD3- CD19- CD56- CD11b- CD117+

Basophil

CD45+ CD3- CD19- CD56- CD11b- FCεRIα+

Eosinophil

CD45+ CD3- CD19- CD56- CD11b+ CD49d+ CD16-

Neutrophil

CD45+ CD3- CD19- CD56- CD11b+ CD49d- CD16+

Mouse

Cell Type

Major Markers

Lineage

T cell

CD45+ CD3+ CD4+ CD8+

Lymphoid

B cell

CD45+ CD45R+

NK cell

CD45+ CD3- CD45R- CD49b+

Monocyte

CD45+ CD3- CD45R- Ly6C+ CD11b+

Myeloid

Macrophage

CD45+ CD3- CD45R- CD11b+ CD68+

DC

CD45+ CD3- CD11c+ CD11b+/-

Mast cell

CD45+ CD3- CD45R- CD11b- CD117+

Basophil

CD45+ CD3- CD45R- CD11b- FCεRIα+

Eosinophil

CD45+ CD3- CD45R- CD11b+ F4/80+ Ly6G-

Neutrophil

CD45+ CD3- CD45R- CD11b+ F4/80- Ly6G+

Investigator can choose different markers in this panel (lineage markers are in gray) to identify major immune cell lineages. Up to 20 colors including viability dye can be tested at one time!

T cell subsets can be characterized based on their effector/memory differentiation status. Upon activation, naive T cells (TN), can proliferate and differentiate into T stem-cell-like memory cells (TSCM), T central memory cells (TCM), T transitional memory cells (TTM), T effector memory cells (TEM), T effector memory cell re-expressing CD45RA (TEMRA), and T terminal effector cells (TTE). The precise identification of these subsets is important for both basic and clinical immune monitoring of patients undergoing T cell-based immunotherapy. Multiple markers are needed to subset the cells to characterize. In this panel, seven distinct CD4 and CD8 T cell populations within the naive and effector cell subsets can be characterized by multiple surface marker staining. ​


Human

Subtypes

Lineage

CD45RA

CD45RO

CD27

CD28

CCR7

CD62L

CD95

TN

CD45+CD3+CD4+
or
CD45+CD3+CD8+

+

-

+

+

+

+

-

TSCM

+

-

+

+

+

+

+

TCM

-

+

+

+

+

+

+

TTM

-

+

+

+

-

-

+

TEM

-

+

-

-

-

-

+

TEMRA

+

+

-

-

-

-

+

TTE

+

-

-

-

-

-

+

Mouse

Subtypes

Lineage

CD45RA

CD45RO

CD27

CD28

CCR7

CD62L

CD95

TN

CD45+CD3+CD4+
or
CD45+CD3+CD8+

-

+

+

+

+

+

-

TSCM

-

+

+

+

+

+

+

TTM

+

+

+

+

+

+

+

TEM

+

-

-

-

-

-

+

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of T cells.

The CD4+ T helper (Th) cells can be further characterized into different sub-types based on cell surface markers, cytokine secretion profile, and transcription factor expression. With this panel, Th1, Th2, Th9, Th17, Th22, and T follicular helper cells (Tfh) populations can be identified. Staining includes cell surface and intracellular staining (ICS) of major markers.​


Human

Subtypes

Lineage

Cell Surface Markers

Secreted Cytokines

Th1

CD45+CD3+CD4+

CD183(CXCR3)+

IFN-γ+ / IL-2+ / TNF-α+ / TNF-
β+

Th2

CD194(CCR4)+ / CD294(CRTH2)+

IL-4+ / IL-5+

Th9

IL-9+

Th17

CD194(CCR4)+ / CD196(CCR6)+

IL-17+ / IL-21+ / IL-22+

Th22

CD194(CCR4)+ / CD196(CCR6)+
/CCR10+

IL-21+ / IL-22+ / CCL-7(MCP-
3)+

Tfh

CD185(CXCR5)+ / PD-1+ / ICOS+

CXCL13+ / IL-4+ / IL-21+ / IFN-γ+

Mouse

Subtypes

Lineage

Cell Surface Markers

Secreted Cytokines

Th1

CD45+CD3+CD4+

CD183(CXCR3)+

IFN-γ+ / IL-2+ / TNF-α+ / TNF-
β+

Th2

CD194(CCR4)+ / CD294(CRTH2)+

IL-4+ / IL-5+

Th9

IL-10+

Th17

CD194(CCR4)+ / CD196(CCR6)+

IL-17+ / IL-21+ / IL-22+

Th22

CD194(CCR4)+ / CD196(CCR6)+
/CCR10+

IL-21+ / IL-22+ / CCL-7(MCP-
3)+

Tfh

CD185(CXCR5)+ / PD-1+ / ICOS+

CXCL13+ / IL-4+ / IL-21+ / IFN-γ+

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of Th cells.

Regulatory T cells (Tregs) are a subset of lymphocytes that can suppress immune responses to maintain immune homeostasis and self-tolerance. Tregs are commonly identified as CD3+ CD4+ CD25+ FoxP3+ CD127low cells in both mice and humans. Several other phenotypical markers can be added, such as cytotoxic T lymphocyte associated‐antigen 4 (CTLA‐4), integrin αE (CD103), programmed death‐1 (PD‐1), lymphocyte‐activation gene 3 (LAG‐3), to further characterize Tregs. ​

Human & Mouse

Subtypes

Lineage

Flow cytometry panel design

Human


CD45+CD3+CD4+


CD25+ / FOXP3+ / CD127low

Mouse

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of T cells.

B cells are a population of the adaptive immune response and target specific antigens using their receptors (antibodies). Naïve B cells leave the bone marrow and migrate to the spleen. In the spleen, they differentiate into different B cell subtypes including transitional B cells, marginal zone B cells, and follicular B cells, which can be further differentiate into terminally differentiated plasma B cells or memory B cells. In this panel, different subtypes of B cells can be identified by staining of major cell surface markers as listed​


Human

Subtypes

Lineage

Cell Surface Markers

Transitional

CD45+CD19+
or
CD45+CD20+

CD24+ / CD27-/ CD38high

Follicular

CD21+ / CD22+ / CD23+/ IgDhigh / IgMlow

Marginal

CD1c+ / CD21+ / CD23- / CD24+ / IgDlow / IgMhigh

Memory

CD27+ / CD38- / CD80+ / CD86+

Plasma

CD45+CD19- or CD45+CD20-

BCMA+ / CD27high / CD38high / CD78+/ CD138+ / CD319+

Mouse

Subtypes

Lineage

Cell Surface Markers

Transitional

CD45+CD45R(B220)+

TACI+ / CD24high / CD93+/ IgM+

Follicular

CD1dlow / CD22+ / CD23+/ CD38+/ IgDhigh / IgMlow

Marginal

CD1d+ / CD9+ / CD21+ / CD22+ / CD35+ / IgDlow / IgMhigh

Memory

CD19+ / CD73+ / CD80+ / CD273(PD-L2)+

Plasma

CD45+CD45R(B220)low

BCMA+ / CD19low / CD27high / CD98+/ Ly6k+

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of B cells.

Natural killer (NK) cells belong to the innate immune system. In humans, they are generally defined as CD45+ CD3- CD56+ cells. In mice, they are generally defined as CD45+CD3-NK1.1+ or NKp46+ cells depending on the mouse strain. While not often evaluated, several immune checkpoint proteins, such as PD-1 (CD279), TIGIT, TIM-3, and VISTA, can be expressed on activated NK cells and can be included in NK cell phenotyping panel.​


Human

Subtypes

Lineage

Cell Surface Markers

CD56highCD16-

CD45+CD3-CD56(NCAM-1)+

CD56high/ CD16- / CD197(CCR7)+ / CD127(IL-7 Rα)-

CD56lowCD16+

CD56low/ CD16+ / CD197(CCR7)- / CD127(IL-7 Rα)-

Mouse

Subtypes

Lineage

Cell Surface Markers

CD11lowCD27high

CD45+CD3-NK1.1+
or
CD45+CD3-NKp46+

CD49b(ITGA2)+/ CD11b(ITGAM)low/ CD27high

CD11highCD27low

CD49b(ITGA2)+ / CD11b(ITGAM)high/ CD27low

CD11highCD27high

CD49b(ITGA2)+ / CD11b(ITGAM)high/ CD27high

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of NK cells.

Dendritic cells (DC) are a heterogeneous population of cell. Two subtypes of DCs have been well described, conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC). The cDC are known to be specialized for absorbing antigens for presentation to naive T cells in adaptive immunity, which are further categorized into two major subpopulations, cDC1 and cDC2. Inflammatory dendritic cells (iDC) are not present in steady-state tissues or lymphoid organs. They differentiate from blood monocytes at sites of inflammation and are therefore frequently called monocyte-derived dendritic cells. In this panel, different subtypes of DCs can be identified by both cell surface staining and intracellular staining (ICS) of major markers (see the following indicated markers).


Human

Subtypes

Lineage

Cell Surface Markers

Secreted
Cytokines

cDC1

CD45+CD3-CD19-
CD14-CD56-

CD141(BDCA-3)+ / XCR1+/ CD11c+ / CLEC9a+ /
IGSF4A+ / CD1a-

IL-12+ / IFN-β+

cDC2

CD1c(BDCA-1)+ / CD11c+ / HLA-DR+/ CD1a-

IL-6+ / IL-1β+ / IL-
12+ / TNF-α+

pDC

CD11clow / CD123(IL-3 Rα)+ / DLEC(BDCA-2)+/
Neuropilin-1(BDCA-4)+ / TLR7+/ TLR9+/ CD1a-

IFN-α+ / IFN-β+ /
IL-6+ / TNF-α+

iDC

CD45+CD3-CD19-
CD56-

CD14+ / CD1a+ / CD1c(BDCA-1)+ / HLA-DR+/
MMR(CD206)+/ SIRPα(CD172a)+

IL-23+ / iNOS+ /
TNF-α+

Mouse

Subtypes

Lineage

Cell Surface Markers

Secreted
Cytokines

cDC1

CD45+CD3-CD4-

CD8α+ / CD11c+ / CLEC9a+ / F4/80- / IGSF4A+/
CD205(DEC-205)+ / CD209(DC-SIGN)- / XCR1+/
MHC class II+ / SIRPα(CD172a)-

IFN-ϒ+ / IL-12+

cDC2

CD45+CD3-CD4+/-

CD11b(ITGAM)+ / CD11c+ / CLEC9a- / F4/80+ /
IGSF4A-/ XCR1- / MHC class II+/ SIRPα(CD172a)+

IFN-ϒ+ / IL-2+
/ IL-6+

pDC

CD45+CD3-
B220/CD45R+

CD11c+ / CLEC9a+ / CD209(DC-SIGN)+ /
CD11b(ITGAM)- / MHC class II+ / Ly6C+/ TLR7+/ TLR9+

IFN-α+ / IFN-
β+ / IL-6+ /
TNF-α+/ IL-12+

iDC

CD45+CD3-CD4-

CD209(DC-SIGN)+ / CD11b(ITGAM)+ / MHC class II+ /
Ly6C+

IL-12+ / TNF-
α+

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of DCs.

Monocytes are derived from macrophage-dendritic cell precursors in the bone marrow and then migrate to the circulating blood and lymphatic system. They are highly heterogeneous and can rapidly change phenotypes based on environmental stimuli. In response to various stimuli, monocytes can migrate to sites of inflammation where they can differentiate into either inflammatory macrophages or monocyte-derived dendritic cells. There are at least two subsets of functionally different monocytes known as inflammatory and patrolling monocytes in both human and mice. In this panel, different subtypes of monocytes can be identified by staining of different markers.​


Human

Subtypes

CD14

CD16

Ly6C

CD62L

CCR2

CX3CR1

Classical

high

-

+

high

-

Non-classical

+

high

-

-

high

Mouse

Subtypes

Ly6C

CD62L

CCR2

CX3CR1

CD49b

Classical

high

+

high

low

+

Non-classical

low

-

-

high

-

Investigator can choose different markers in this panel to identify subtypes of monocytes.

Macrophages are a group of highly heterogeneous cell population that can rapidly change their phenotypes and functional properties based on environmental stimuli. They are not only involved in the recognition, phagocytosis, and degradation of cellular debris and pathogens, but also in maintaining tissue integrity and homeostasis. In general, macrophages can be classified into classically activated M1 macrophages and alternatively activated M2 macrophages based on their function and activation. In response to different stimulation, M2 macrophage can be further divided into the M2a, M2b, M2c, and M2d subtypes. In this panel, different types of macrophages can be identified by staining of certain cell surface and intracellular markers.​


Human

Subtypes

Cell Surface Markers

Secreted Cytokines

General

CD11b(ITGAM) / CD14 / CD68 / CD16(FCϒRIII) / CD64(FCϒRI) /
CCR5 / EMR1

M1

CD68+ / CD80+ / CD86+ / CD163low / HLA-DRhigh

IL-1β+ / IL-6+ / IL-12+
/
IL-23+ / TNF-α+/ IFN-
γ+

M2a

CD14low / CD163low / TLR4low / CD200R+ / CD206high / HLA-DRlow

IL-1ra+ / IL-10+ / IL-
13+ /
IL-14+ / TGF-β+

M2b

CD14high / CD80high / CD200Rlow / HLA-DR+

IL-1β+ / IL-6+ / IL-10+
/
TNF-α+

M2c

CD86low / CD163high / CD206+ / TLR1+ / CCR2+

IL-10+ / TGF-β+

Mouse

Subtypes

Cell Surface Markers

Secreted Cytokines

General

CD11b(ITGAM) / CD14 / CD68 / CD16(FCϒRIII) / CD64(FCϒRI) /
CCR5 / F4/80

M1

CD68+ / CD80+ / CD86+ / CD163low / MHC class IIhigh

IL-1β+ / IL-6+ / IL-12+
/
IL-23+ / TNF-α+/ IFN-
γ+

M2a

CD14low / CD163low / TLR4low / CD200R+ / CD206high/ MHC
class IIlow

IL-1ra+ / IL-10+ / IL-
13+ /
IL-14+ / TGF-β+

M2b

CD14high / CD80high / CD200Rlow / MHC class II+

IL-1β+ / IL-6+ / IL-10+
/
TNF-α+

M2c

CD86low / CD163high / CD206+ / TLR1+ / CCR2+

IL-10+ / TGF-β+

Investigator can choose different markers in this panel to identify subtypes of macrophages. ​

Myeloid-derived suppressor cells (MDSC) are a group of myeloid cells that can suppress immune responses. MDSC levels are negatively correlated with prognosis and overall survival of cancer patients. There are two main subsets of MDSCs, the monocytic (M-MDSC) and the granulocytic (G-MDSC), that are defined by their surface phenotypes. However, multiple human MDSC subsets with different phenotypes have been documented in several types of tumors. Therefore, more surface markers and staining may be required to fully characterize the subsets, origin, and function of MDSCs in your sample.


Human

Subtypes

Lineage

Cell Surface Markers

M-MDSC

CD45+CD3-CD19-CD56-

CD11b+ / CD14+ / HLA-DR-/low

G-MDSC

CD11b+ /CD15+ / CD14- / CD33+/low /CD66b+

Mouse

Subtypes

Lineage

Cell Surface Markers

M-MDSC

CD45+CD3-CD19-CD56-

CD11b+ / Ly6G- / Ly6Chi

G-MDSC

CD11b+ / Ly6G+ / Ly6Clo

Investigator can choose lineage plus any other markers in this panel to identify different subtypes of MDSCs.

Granulocytes are the most common type of leukocytes. Thy have multilobed nuclei and small, enzyme-containing granules in their cytoplasm. They migrate to the site of inflammation and release several different chemicals and enzymes to fight infection as part of the immune response. There are four types of granulocytes: neutrophils, eosinophils, basophils, and mast cells. This panel helps to identify different types of granulocytes by staining of major markers.


Human

Subtypes

Cell Surface Markers

Basophils

FCεRIα+ / CRTH-2+/ CD49b(ITGA2)+/ CD69+/ CD117(c-kit)-/ CD123(IL-3 Rα)high /
CD193(CCR3)+/ CD294(CRTH2)+

Eosinophils

Siglec-8+ / CD11b(ITGAM)+ / CD14-/low / CD15+ / CD16(FCγRIII)-/ CD49d(ITGA4)+/
CD125(IL-5 Rα)+/ CD193(CCR3)+/ CD294(CRTH2)+

Neutrophils

CD11b(ITGAM)+ / CD14-/low / CD15+ / CD16(FCγRIII)+/ CD32(FCγRII)+/ CD33(Siglec-3)+ /
CD49d(ITGA4)-/ CD66b(CEACAM)+

Mast Cells

FCεRIα+ / CD11b(ITGAM)-/ CD11c-/low / CD32+ / CD33(Siglec-3)+ / CD49b(ITGA2)-/
CD117(c-kit)high/ CD123(IL-3 Rα)low/+ / CD203c(ENPP-3)+

Mouse

Subtypes

Cell Surface Markers

Basophils

FCεRIα+ / CD294(CRTH2)+ / CD49b(ITGA2)+/ CD69+/ CD117(c-kit)- / CD123(IL-3 Rα)high /
CD193(CCR3)+

Eosinophils

Siglec-F+ / F4/80+ / Ly-6G- / CD11b(ITGAM)+ / CD16(FCγRIII)-/ CD49d(ITGA4)+/
CD115(M-CSFR)-/ CD125(IL-5Rα)+ / CD193(CCR3)+

Neutrophils

Siglec-F- / F4/80- / Ly-6G+ / CD11b(ITGAM)+ / CD18+ / CD115(M-CSFR)-

Mast Cells

FCεRIα+ / CD11b(ITGAM)-/ CD11c-/low / CD34+ / CD49b(ITGA2)- / CD117(c-kit)high /
CD123(IL-3 Rα)low/+ / CD203c(ENPP-3)+

Investigator can choose different markers in this panel to identify subtypes of granulocytes.

Stem cells are undifferentiated or partially differentiated cells able to differentiate into different cell types, and which are identified by the presence and absence of certain cell surface markers.​
Hematopoietic stem cells (HSCs) are stem cells that can be differentiated into other blood cells and can be found in bone marrow and peripheral blood.​
Mesenchymal stem cells (MSCs), also known as multi-potent mesenchymal stromal cells, are adult stem cells that are found not only in bone marrow, but also in different human organs such as adipose tissue, as well as in the umbilical cord. These can be differentiated into a variety of cell types including osteoblasts, chondrocytes, adipocytes, and myocytes.​

Cell
types

Cell Surface Markers

HSCs

Lin- / CD34+/ CD38-/ CD45RA-/ CD90+/ CD49f+



MSCs

Positive

CD29 / CD44 / CD51 / CD73 / CD82 / CD90 / CD105 / CD106 / CD166 / FSP-1
(anti-human S100A4 antibody)

Negative

CD14 /CD11b / CD19 / CD31 / CD34 / CD45 / CD79α / CD117 / CD133 / HLA-DR

Investigator can choose different markers in this panel to identify HSCs or MSCs.