Nexcelom Bioscience
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Introduction to Surface and Intracellular Proteins Markers

Surface and intracellular proteins are markers that are important for identifying specific type of cells and characterizing cell populations. Traditionally, these assays are performed on flow cytometers. In the recent years, image-based cytometry method has been utilized for many standard surface and intracellular protein detection assay to provide a more simple and rapid method for cell population analysis. The Cellometer Vision CBA image cytometer is specifically designed to perform surface marker and intracellular protein fluorescence analysis to characterize cell populations. Below are examples of some of the assays have been performed using the Cellometer image cytometry method.

Immunophenotyping for Immunological Research

Biomarkers related to surface protein express on various types of immune cells such as T Cell, Activated T Cell, B Cell, Lymphocytes, splenocytes, thymocytes, etc. can be detected and measured on the Cellometer Vision CBA image cytometer. The data shown below are some examples of fluorescent intensity data measured from bright field and fluorescent images captured by the Cellometer Vision CBA image cytometry method. These biomarkers are important to characterize cell samples for downstream assays such as immune cell purification, activated immune cell populations, and cell sorting.

Human Activated T Cell – CD44-APC

Human T-cells were stained with APC conjugated anti-CD44 and imaged on the Cellometer Vision CBA. The counted bright field image shows T-cells automatically identified by the Cellometer software and outlined in green color. The fluorescent APC image shows red surface marker staining of positively stained T-cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that more than 95% of the cells were positive for CD44.

human activated T cells - CD44-APC

Bright Field Counted Image

Bright field counted cell image of human activated T cells

Fluorescence Image

Fluorescence cell image of human activated T cells

Human Activated T Cell – CD44-PE

Similarly to the experiment above, Human T-cells were stained with phycoerythrin (PE) conjugated anti-CD44 and imaged on the Cellometer Vision CBA. The counted bright field image shows T-cells automatically identified by the Cellometer software and outlined in green color. The fluorescent PE image shows orange surface marker staining of positively stained T-cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that more than 95% of the cells were positive for CD44.

Human Activated T Cell – CD44-PE

Bright Field Counted Image

Bright field counted cell image of Human Activated T Cell – CD44-PE

Fluorescence Image

Fluorescence cell image of Human Activated T Cell – CD44-PE

Macrophage in Mouse Bone Marrow – CD11b-PE

Primary bone marrow sample was stained with PE conjugated anti-CD11b, a cell surface receptor found on the surface of several cells types including mononuclear phagocytes a.k.a macrophages. Twenty microliters of the stained sample was imaged on the Cellometer Vision CBA system. The green circles shown in the bright field image represent automatically counted and outlined mononuclear cells. The fluorescent image shows bright staining profile of cells that are positive for CD11b. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that about 84% of the cells are positive for CD11b.

Macrophage in Mouse Bone Marrow – CD11b-PE

Bright Field Counted Image

Bright field counted cell image of Macrophage in Mouse Bone Marrow – CD11b-PE

Fluorescence Image

Fluorescence cell image of Macrophage in Mouse Bone Marrow – CD11b-PE

Human Jurkat Cells – CD3-PE

Cultured Jurkat cells were stained with PE conjugated anti-CD3 surface marker and imaged on the Cellometer Vision CBA. The counted bright field image shows Jurkat cells that were automatically identified by the Cellometer software and outlined in green color. The fluorescent PE image shows orange surface marker staining of CD3 positive Jurkat cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that more than 80% of the cells were positive for CD3.

Human Jurkat Cells – CD3-PE

Bright Field Counted Image

Bright field counted cell image of Human Jurkat Cells – CD3-PE

Fluorescence Image

Fluorescence cell image of Human Jurkat Cells – CD3-PE

Human PBMC – CD3-FITC

Primary peripheral blood mononuclear cell (PBMC) sample was stained with FITC conjugated anti-CD3 surface marker and imaged on the Cellometer Vision CBA. The counted bright field image shows cells that were automatically identified by the Cellometer software and outlined in green color. The fluorescent FITC image shows green surface marker staining of CD3 positive mononuclear cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that nearly 90% of the cells were positive for CD3.

Human PBMC – CD3-FITC

Bright Field Counted Image

Bright field counted cell image of Human PBMC – CD3-FITC

Fluorescence Image

Fluorescence cell image of Human PBMC – CD3-FITC

Human PBMCs – CD3-PECy5

Similarly to the CD3-FITC experiment above, primary peripheral blood mononuclear cell (PBMC) sample was stained with anti-CD3 PE-Cy5 conjugated tandem dye and imaged on the Cellometer Vision CBA. Please note that this tandem dye is a combination of phycoerythrin (PE) and cyanine (Cy5), has a broad emission spectra and was imaged using the APC filter module. The counted bright field image shows cells that were automatically identified by the Cellometer software and outlined in green color. The fluorescent image shows a reddish surface marker staining of positively stained mononuclear cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that about 65% of the cells were positive for CD3.

Human PBMCs – CD3-PECy5

Bright Field Counted Image

Bright field counted cell image of Human PBMCs – CD3-PECy5

Fluorescence Image

Fluorescence cell image of Human PBMCs – CD3-PECy5

Human PBMCs – CD19-PECy5

Primary peripheral blood mononuclear cell (PBMC) sample was stained with anti-CD19 PE-Cy5 conjugated tandem dye and imaged on the Cellometer Vision CBA. Please note that this tandem dye is a combination of phycoerythrin (PE) and cyanine (Cy5), has a broad emission spectra and was imaged using the APC filter module. The counted bright field image shows cells that were automatically identified by the Cellometer software and outlined in green color. The fluorescent image shows a reddish surface marker staining of positively stained B-cells. The mean fluorescent intensity values for each cell were exported to flow cytometry software FCS express and plotted as a histogram. The gated data shows that about 10% of the cells were positive for CD19 B-cells.

Human PBMCs – CD19-PECy5

Bright Field Counted Image

Bright field counted cell image of Human PBMCs – CD19-PECy5

Fluorescence Image

Fluorescence cell image of Human PBMCs – CD19-PECy5

Mouse Cells – Mouse IgG-FITC and RFP

Mouse Cells – Mouse IgG-FITC and RFP scatter plot

Bright Field Counted Image

Bright field counted cell image of Mouse IgG-FITC and RFP

Fluorescence Image

Fluorescence cell image of Mouse IgG-FITC and RFP

Fluorescence Image

Fluorescence cell image of Mouse IgG-FITC and RFP

CD44 - PE

CD44 - PE

Bright Field Counted Image

Bright field counted cell image of CD44 - PE

Fluorescence Image

Fluorescence cell image of CD44 - PE

Human Lymphocytes – CD4-PE

Human Lymphocytes – CD4-PE

Mouse Thymocytes – CD8-PE and CD4-APC

Mouse Thymocytes – CD8-PE and CD4-APC

Mouse Splenocytes – CD8-PE and CD4-APC

Mouse Splenocytes – CD8-PE and CD4-APC

MHC Dextramer – CD8-FITC and Dextramer-PE

MHC Dextramer – CD8-FITC
MHC Dextramer – CD8-FITC
MHC Dextramer – Dextramer-PE

Human T Cells and Cytokines - CD8-PE and INFg-FITC

Human T Cells and Cytokines -  CD8-PE and INFg-FITC

Intracellular Protein for Protein Pathway Research

Various complex proteins exist in the cells that are generated or removed by cellular functions. These proteins can be an indicator to measure certain protein pathways or functions of the cells to determine if they are operating normally or abnormally. These intracellular proteins can be fluorescently labeled and measured by Cellometer Vision CBA image cytometer to determine the cell population associated with these proteins.

Heat Shock Protein in MNNG Cancer Cells – HSP90-PE and GFP

Heat Shock Protein in MNNG Cancer Cells – HSP90-PE and GFP

Bright Field Counted Image

Bright field counted cell image of Heat Shock Protein in MNNG Cancer Cells

Fluorescence Image

Fluorescence cell image of Heat Shock Protein in MNNG Cancer Cells

Fluorescence Image

Fluorescence cell image of Heat Shock Protein in MNNG Cancer Cells

Heat Shock Protein in MNNG Cancer Cells – Grp78-Alexa Fluor 594 and GFP

Heat Shock Protein in MNNG Cancer Cells – Grp78-Alexa Fluor 594 and GFP

Bright Field Counted Image

Bright field counted cell image of Heat Shock Protein in MNNG Cancer Cells

Fluorescence Image

Fluorescence cell image of Heat Shock Protein in MNNG Cancer Cells

Fluorescence Image

Fluorescence cell image of Heat Shock Protein in MNNG Cancer Cells

Stem Cell Analysis for Regenerative Medicine

Stem cell markers are often used to determine the stem cell population in blood, cord blood, bone marrow, and adipose tissue. These cells are extremely important for research in regenerative medicine, and the ability to quickly characterize a stem cell population in a cell sample can greatly improve research efficiency. Cellometer Vision CBA image cytometer can be utilized to measure cell population of some of the common stem cell markers.

Mouse iPS Cells – SSEA-1-PE

Mouse iPS Cells – SSEA-1-PE

Bright Field Counted Image

Bright field counted cell image of Mouse iPS Cells – SSEA-1-PE

Fluorescence Image

Fluorescence cell image of Mouse iPS Cells – SSEA-1-PE

Human iPS Cells – SSEA-4-PE

Human iPS Cells – SSEA-4-PE

Bright Field Counted Image

Bright field counted cell image of Human iPS Cells – SSEA-4-PE

Fluorescence Image

Fluorescence cell image of Human iPS Cells – SSEA-4-PE

Human iPS Cells – TRA-181-PE

Human iPS Cells – TRA-181-PE

Bright Field Counted Image

Bright field counted cell image of Human iPS Cells – TRA-181-PE

Fluorescence Image

Fluorescence cell image of Human iPS Cells – TRA-181-PE

CACO2 Cancer Stem Cells – CD133-FITC

CACO2 Cancer Stem Cells – CD133-FITC

Bright Field Counted Image

Bright field counted cell image of CACO2 Cancer Stem Cells – CD133-FITC

Fluorescence Image

Fluorescence cell image of CACO2 Cancer Stem Cells – CD133-FITC
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