Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel
Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel Summary
For the identification and characterization of neural progenitor cells.
Key Benefits
- Eight markers increase the confidence in accurate cell identification
- High quality antibodies provide consistent and reproducible results
- Cost-effective use of resources
Why use a Panel of Antibodies to Identify and Characterize Neural Progenitor Cells?
Neural progenitor cells (NPC) reside in several areas of the embryonic and adult central nervous system and are defined by their ability to self-renew and differentiate into neurons, astrocytes, and oligodendrocytes. Various molecular markers can be used to identify NPC populations in vivo and in vitro.However, since most markers are expressed in a variety of cell types, several markers must be examined to thoroughly define a cell population of interest.
The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel is designed for the characterization of human/mouse/rat NPC marker expression by immunocytochemistry (all markers) and flow cytometry (CXCR4 and SSEA-1). The panel can be used to detect the following marker molecules: CXCR4, Musashi-1, Nestin, Notch-1, SOX1, SOX2, SSEA-1, and Vimentin.
The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel:
- Includes eight antibodies to increase confidence in cell identification and characterization.
- Compatible with detection of SSEA-1 and CXCR4 by flow cytometry, which can be completed in less than two hours.
- Compatible with detection of all markers by immunocytochemistry, which can be completed in less than six hours.
Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025) includes eight antibodies that can be used to detect expression of CXCR4, Musashi, Nestin, Notch-1, SOX1, SOX2, SSEA-1, and Vimentin.
- Mouse Anti-CXCR4 Monoclonal Antibody (clone 44708, isotype mouse IgG2A)
- Goat Anti-Musashi-1 Antigen Affinity-purified Polyclonal Antibody
- Mouse Anti-Nestin Monoclonal Antibody (clone 307501, isotype mouse IgG2A)
- Goat Anti-Notch-1 Antigen Affinity-purified Polyclonal Antibody
- Goat Anti-SOX1 Antigen Affinity-purified Polyclonal Antibody
- Goat Anti-SOX2 Antigen Affinity-purified Polyclonal Antibody
- Mouse Anti-SSEA-1 Monoclonal Antibody (clone MC-480, isotype mouse IgM)
- Rat Anti-Vimentin Monoclonal Antibody (clone 280618, isotype rat IgG2A)
Each antibody is supplied as a 10X stock; enough for 25 assays when used in a 50 µL staining volume per assay.
Stability and Storage
The unopened kit can be stored at 2 °C to 8 °C and should be used within one year of receipt.
The opened/Reconstituted Reagents may be stored for up to 1 month at 2 °C to 8 °C or aliquoted and stored at =-20 °C in a manual defrost freezer for up to 6 months. Avoid repeated freeze-thaw cycles.
PRECAUTION
Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
LIMITATIONS OF THE PROCEDURE
- FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
- The safety and efficacy of this product in diagnostic or other clinical uses have not been established.
Specifications
Product Datasheets
Scientific Data
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Verification of Neural Progenitor Marker Expression in Rat Neural Stem Cells. Rat cortical stem cells (Catalog # NSC001), were assessed for expression of neural progenitor markers using the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025). Rat cortical stem cells were stained withA) a Mouse Anti-CXCR4 Monoclonal Antibody followed by the NorthernLights™(NL)557-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL007; red)B) a Goat Anti-Musashi-1 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001; red), andC) a Mouse Anti-Nestin Monoclonal Antibody followed by the NL493-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL009; green). The nuclei were counterstained with DAPI (blue) in each panel.
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Verification of Neural Progenitor Marker Expression in Mouse Neural Stem Cells. Mouse cortical stem cells (Catalog # NSC002) were assessed for expression of neural progenitor markers using the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025). Mouse cortical stem cells were stained withA)a Mouse Anti-SSEA-1 Monoclonal Antibody followed by the NL557-conjugated Goat Anti-Mouse Secondary Antibody (Catalog # NL019; red),B)a Goat Anti-Notch-1 Antigen Affinity-purified Polyclonal Antibody followed by the NL493-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL003; green), andC) a Goat Anti-SOX2 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001; red). The nuclei were counterstained with DAPI (blue) in each panel.
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Analysis of Human Embryonic Stem Cell Growth and Stemness Post-staining. Human neural stem cells were assessed for expression of neural progenitor markers using the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025). Human neural stem cells were stained with A) a Rat Anti-Vimentin Monoclonal Antibody followed by the NL557-conjugated Goat Anti-Rat Secondary Antibody (Catalog # NL013; red) and B) a Goat Anti-SOX1 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001; red). The nuclei were counterstained with DAPI (blue) in each panel.
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Detection of Neural Progenitor Markers CXCR4 and SSEA-1 by Flow Cytometry. Cells were stained with antibodies included in the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025).A) CXCR4 was detected in undifferentiated Mouse Cortical Stem Cells (Catalog # NSC002) using a Mouse Anti-CXCR4 Monoclonal Antibody (filled histogram) or a mouse IgG2Aisotype control (open histogram).B) SSEA-1 was detected in undifferentiated Rat Cortical Stem Cells (Catalog # NSC001) using a Mouse Anti-SSEA-1 Monoclonal Antibody (filled histogram) or a mouse IgM isotype control (empty histogram). Cells were stained using PE-conjugated secondary developing reagents.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, the expression of neural progenitor cell markers can be assessed by flow cytometry or immunocytochemistry using the following procedure:
- For ICC, fix and label cells with the provided primary antibodies
- For Flow Cytometry, label cells with the provided primary antibodies
- Stain the cells with a fluorochrome-conjugated secondary antibody
- Analyze the samples by immunocytochemistry or flow cytometry
Reagents Included in the Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel (Catalog # SC025):
- Mouse Anti-Nestin Monoclonal Antibody (clone 307501, isotype mouse IgG2A) - 25 µg
- Goat Anti-SOX1 Antigen Affinity-purified Polyclonal Antibody- 25 µg
- Goat Anti-SOX2 Antigen Affinity-purified Polyclonal Antibody- 25 µg
- Rat Anti-Vimentin Monoclonal Antibody (clone 280618, isotype rat IgG2A) - 25 µg
- Goat Anti-Notch-1 Antigen Affinity-purified Polyclonal Antibody - 25 µg
- Goat Anti-Musashi-1 Antigen Affinity-purified Polyclonal Antibody- 25 µg
- Mouse Anti-CXCR4 Monoclonal Antibody (clone 44708, isotype mouse IgG2A) - 25 µg
- Mouse Anti-SSEA-1 Monoclonal Antibody (clone MC-480, isotype mouse IgM) - 25 µg
Reagents
- Flow Cytometry Staining Buffer (Catalog # FC001)
- Sterile PBS
- 1% BSA in PBS
- 4% Paraformaldehyde in PBS
- 10% Normal donkey serum
- Deionized of distilled water
- Triton® X-100
- Isotype Control Antibodies (See Table 1)
- Secondary Developing Reagents (See Table 1)
Materials
- FACS™ Tubes or 5 mL round-bottom polystyrene tubes (flow cytometry tubes)
- Pipettes and pipette tips
Equipment
- Benchtop centrifuge
- 2 °C to 8 °C refrigerator
Table 1: Isotype Controls and Secondary Developing Reagents Required
| Primary Antibody |
Isotype Control |
Secondary Developing |
Secondary Developing |
| Nestin | NorthernLights conjugated Donkey Anti- Mouse IgG (Catalog # NL007, NL008, or NL009) |
||
| SOX1 SOX2 Musashi-1 Notch-1 |
NorthernLights conjugated Donkey Anti- Goat IgG (Catalog # NL001, NL002, or NL003) |
||
| Vimentin | NorthernLights conjugated Goat Anti- Rat IgG (Catalog # NL013, NL014, or NL015 |
||
| CXCR4 | Mouse IgG2A (Catalog # MAB003) | NorthernLights conjugated Donkey Anti- Mouse IgG (Catalog # NL007, NL008, or NL009) |
Goat Anti-Mouse IgG (Catalog # F0101B, F0102B, F0103B, or F0114) |
| SSEA-1 | Mouse IgM | NorthernLights conjugated Goat Anti- Mouse IgM (Catalog # NL019 or NL020) |
Goat Anti-Mouse IgM (Catalog # F0116, F0117, F0118, or F0119) |
Characterization of Neural Progenitor Cell Markers by Immunocytochemistry
Coat coverslips with Poly-L-ornithine and Fibronectin or a defined matrix.
Plate neural progenitor cells.
Culture cells to the desired confluency.
Fix stem cells with 4% paraformaldehyde.
Block in blocking solution containing 0.3% Triton X-100.
Incubate with reconstituted primary antibodies.
Wash with wash buffer.
Incubate with fluorochrome-conjugated secondary developing reagent.
Wash with wash buffer.
Incubate with nuclear counterstain
Mount the coverslip.
Visualize using a fluorescence microscope and appropriate filter sets.
Characterization of Neural Progenitor Cell Markers SSEA-1 and CXCR4 by Flow Cytometry
Perform a cell count on harvested cells.
Resuspend cells in Flow Cytometry Staining Buffer.
Aliquot 90 µL of the cells into 5 mL flow cytometry tubes.
Add 10 µL of antibody or isotype control (or a previously titrated amount).
Vortex and incubate for 30 minutes at room temperature.
Centrifuge samples at 300 x g for 5 minutes.
Wash the samples three times with Flow Cytometry Staining Buffer.
Resuspend each sample in 200 μL of Flow Cytometry Staining Buffer.
Add 10 µL of a fluorochrome-conjugated secondary antibody (or a previously titrated amount).
Incubate for 30 minutes at room temperature in the dark.
Centrifuge the samples at 300 x g for 5 minutes.
Wash the samples with Flow Cytometry Staining Buffer.
Resuspend the cells in 200-400 µL of Flow Cytometry Staining Buffer
Analyze the cells by flow cytometry.
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