Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel

Transcription

1 Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel Catalog Number SC025 Reagents for the identification of human/mouse/rat neural progenitor cells. This package insert must be read in its entirety before using this product. For laboratory research use only. Not for diagnostic use. The safety and efficacy of this product in diagnostic or other clinical uses has not been established.

2 TABLE OF CONTENTS SECTION PAGE INTRODUCTION...1 DESIGN OF THE PANEL...1 LIMITATIONS OF THE PROCEDURE...1 PRECAUTION...1 MATERIALS PROVIDED...2 OTHER SUPPLIES REQUIRED...3 REAGENT PREPARATION...4 PROCEDURE...4 DATA EXAMPLES...6 RELATED REAGENTS...8 REFERENCES...9 MANUFACTURED AND DISTRIBUTED BY: USA & Canada R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413, USA TEL: (800) (612) FAX: (612) DISTRIBUTED BY: UK & Europe R&D Systems Europe, Ltd. 19 Barton Lane, Abingdon Science Park, Abingdon OX14 3NB, UK TEL: +44 (0) FAX: +44 (0) China R&D Systems China Co., Ltd. 24A1 Hua Min Empire Plaza, 726 West Yan An Road, Shanghai PRC TEL: +86 (21) FAX: +86 (21)

3 INTRODUCTION Neural progenitor (NP) cells reside in several areas of the embryonic and adult central nervous system and can be harvested and propagated in vitro. NP cells are defined by their ability to self renew and differentiate into neurons, astrocytes, and oligodendrocytes (1, 2). Various molecular markers can facilitate identification of NP cell populations in vivo and in vitro. Due to the wide expression of most markers in a variety of cell types, it is important to look at several markers to ensure you have the population of interest. The intermediate filament protein, Nestin, has been shown to identify stem/progenitor cells in the developing nervous system (3). Nestin is now used frequently as a marker for NP cells both in vivo and in vitro. Another intermediate filament protein, Vimentin, is expressed in the developing neural tube, marking progenitor cells (4). Musashi-1 is an RNA binding protein essential for NP cell self renewal by translational inhibition of its target genes, one of which is Numb. Numb is an inhibitor of Notch signaling, which is important for NP cell proliferation (5). The SRY-box transcription factors SOX1 and SOX2 are expressed in NP cells of the embryo and adult. Both of these proteins have been used to specifically sort NP cells from surrounding tissue, and are thus considered important markers for this cell population (6, 7). Cell surface markers for stem cell populations are valuable in cell sorting progenitor cells from more differentiated phenotypes. CXCR4 and SSEA-1 are expressed on NP cells (8-10). CXCR4 plays a role in NP cell migration during injury (7, 8). SSEA-1 is a useful marker for identification and sorting of NP cells from the adult brain (10). DESIGN OF THE PANEL The Human/Mouse/Rat Neural Progenitor Cell Marker Antibody Panel is designed for the identification and characterization of human/mouse/rat neural progenitor cells by marker expression. The panel contains the following antibodies: Anti-Nestin, Anti-SOX1, Anti-SOX2, Anti-Vimentin, Anti-Notch-1, Anti-Musashi-1, Anti-CXCR4, and Anti-SSEA-1. LIMITATIONS OF THE PROCEDURE FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE. The safety and efficacy of this product in diagnostic or other clinical uses has not been established. PRECAUTION The acute and chronic effects of overexposure to reagents in this kit are unknown. Safe laboratory procedures should be followed, and protective clothing should be worn when handling kit reagents. 1

4 MATERIALS PROVIDED Store unopened kit at 2-8 C in a manual defrost freezer. This kit is stable for up to one year from the date of receipt. PART PART # DESCRIPTION Anti-Nestin Purified Mouse Monoclonal IgG 2A Clone: Anti-SOX1 Affinity Purified Goat IgG Anti-SOX2 Affinity Purified Goat IgG Anti-Vimentin Purified Rat Monoclonal IgG 2A Clone: Anti-Notch-1 Affinity Purified Goat IgG Anti-Musashi-1 Affinity Purified Goat IgG Anti-CXCR4 Purified Mouse Monoclonal IgG 2A Clone: Anti-SSEA-1 Purified Mouse Monoclonal IgM Clone: MC *Provided this is within one year from the date of receipt. 25 μg of a monoclonal antibody specific for Nestin; lyophilized. 25 μg of a polyclonal antibody specific for SOX1; lyophilized. 25 μg of a polyclonal antibody specific for SOX2; lyophilized. 25 μg of a monoclonal antibody specific for Vimentin; lyophilized. 25 μg of a polyclonal antibody specific for Notch-1; lyophilized. 25 μg of a polyclonal antibody specific for Musashi-1; lyophilized. 25 μg of a monoclonal antibody specific for CXCR4; lyophilized. 25 μg of a monoclonal antibody specific for SSEA-1; lyophilized. STORAGE OF OPENED/ RECONSTITUTED MATERIAL May be stored for up to 1 month at 2-8 C or aliquoted and stored at -20 C in a manual defrost freezer for up to 6 months.* Avoid repeated freeze-thaw cycles. 2 For research use only. Not for use in diagnostic procedures.

5 OTHER SUPPLIES REQUIRED Flow Cytometry Staining Buffer (R&D Systems, Catalog # FC001) Sterile PBS 1% BSA in PBS 4% paraformaldehyde in PBS 10% Normal donkey serum Deionized or distilled water Triton TM X well tissue culture plate Coverslips (Sterilized) Fluorescence microscope Microscope slide Benchtop centrifuge 2-8 C refrigerator Additional supplies required are as follows: PRIMARY ANTIBODY ISOTYPE CONTROL SECONDARY DEVELOPING REAGENTS FOR IMMUNOCYTOCHEMISTRY SECONDARY DEVELOPING REAGENTS FOR FLOW CYTOMETRY Nestin NorthernLights conjugated Donkey Anti-Mouse IgG (R&D Systems, Catalog # NL007, NL008, or NL009) SOX1 SOX2 Musashi-1 Notch-1 NorthernLights conjugated Donkey Anti-Goat IgG (R&D Systems, Catalog # NL001, NL002, or NL003) Vimentin NorthernLights conjugated Goat Anti-Rat IgG (R&D Systems, Catalog # NL013, NL014, or NL015) CXCR4 Mouse IgG 2A (R&D Systems, Catalog # MAB003) NorthernLights conjugated Donkey Anti-Mouse IgG (R&D Systems, Catalog # NL007, NL008, or NL009) Goat Anti-Mouse IgG (R&D Systems, Catalog # F0101B, F0102B, F0103B, or F0114) SSEA-1 Mouse IgM NorthernLights conjugated Goat Anti-Mouse IgM (R&D Systems, Catalog # NL019 or NL020) Goat Anti-Mouse IgM (R&D Systems, Catalog # F0116, F0117, F0118, or F0119) 3

6 REAGENT PREPARATION Reconstitute each vial with 250 μl of sterile PBS. This provides a 10X stock solution. Note: Optimal dilutions should be determined by each laboratory for each application. PROCEDURE Surface Marker Analysis of CXCR4 and SSEA-1 by Flow Cytometry 1. Resuspend the cells in Flow Cytometry Staining Buffer at a concentration of 1 x 10 6 cells/ml. 2. For each marker, transfer 90 μl of the cell suspension into a separate 5 ml tube. Add 10 μl of either Anti-CXCR4 or Anti-SSEA Incubate for 30 minutes at room temperature. 4. Following incubation, wash the sample twice in 2 ml of Flow Cytometry Staining Buffer. 5. Resuspend the cells in 200 μl of Flow Cytometry Staining Buffer and add a secondary developing reagent such as Goat Anti-Mouse IgG or IgM conjugated to a fluorochrome according to the manufacturer's instructions (see the table on page 3). 6. Incubate for 30 minutes at room temperature in the dark. 7. Following incubation, wash the sample twice in 2 ml of Flow Cytometry Staining Buffer. 8. Resuspend the cells in μl of Flow Cytometry Staining Buffer for flow cytometric analysis. Notes: As a control for analysis, cells in a separate tube should be treated with a flow cytometry isotype control (see the table on page 3). Anti-CXCR4 and Anti-SSEA-1 have been tested by flow cytometry using mouse and rat neural progenitors. Human neural progenitors have not been tested by flow cytometry. 4 For research use only. Not for use in diagnostic procedures.

7 PROCEDURE CONTINUED Immunocytochemistry Note: This protocol is for cells grown on coverslips in a 24-well tissue culture plate. 1. Wash the cells twice with 1 ml of sterile PBS. 2. Fix the cells with 0.5 ml of 4% paraformaldehyde in PBS for 20 minutes at room temperature. 3. Wash the cells three times with 0.5 ml of 1% BSA in PBS for 5 minutes. 4. Permeabilize and block the cells with 0.5 ml of 1% BSA in PBS containing 10% normal donkey serum and 0.3% Triton X-100 at room temperature for 45 minutes. 5. While the cells are being blocked, dilute the reconstituted antibody in 1% BSA in PBS containing 10% normal donkey serum and 0.3% Triton X-100 to a final concentration of 10 μg/ml. 6. After blocking, incubate the cells with 300 μl/well of antibody working solution for 3 hours at room temperature or overnight at 2-8 C. Note: A negative control should be run using 1% BSA in PBS containing 10% normal donkey serum and 0.3% Triton X-100 with no primary antibody. 7. Wash the cells three times with 0.5 ml of 1% BSA in PBS for 5 minutes each wash. 8. Dilute the secondary developing reagent 1:200 in 1% BSA in PBS (see the table on page 3). 9. Incubate the cells with 300 μl/well of secondary developing reagent for 60 minutes at room temperature in the dark. 10. Wash the cells three times with 0.5 ml of 1% BSA in PBS for 5 minutes each wash. 11. Aspirate the PBS from the wells and add 0.5 ml of deionized or distilled water. Carefully remove the coverslips with forceps and mount cell side down onto a drop of mounting medium on a large slide. 12. Slides are ready for microscopic observation. 5

8 DATA EXAMPLES A. B. CXCR4/DAPI C. Musashi-1/DAPI Nestin/DAPI Figure 1: Verification of Neural Progenitor Marker Expression in Rat Neural Stem Cells. Rat cortical stem cells (R&D Systems, Catalog # NSC001), were assessed for expression of neural progenitor markers using this kit. Rat cortical stem cells were stained with A. Mouse AntiCXCR4 Monoclonal Antibody followed by the NorthernLights (NL) 557-conjugated Donkey Anti-Mouse Secondary Antibody (R&D Systems, Catalog # NL007; red) B. Goat Anti-Musashi-1 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody (R&D Systems, Catalog # NL001; red), and C. Mouse Anti-Nestin Monoclonal Antibody followed by the NL493-conjugated Donkey Anti-Mouse Secondary Antibody (R&D Systems, Catalog # NL009; green). The nuclei were counterstained with DAPI (blue) in each panel. A. B. SSEA-1/DAPI CXCR4/DAPI C. Musashi-1/DAPI Notch-1/DAPI SOX2/DAPI Figure 2: Verification of Neural Progenitor Marker Expression in Mouse Neural Stem Cells. Mouse cortical stem cells (R&D Systems, Catalog # NSC002) were assessed for expression of neural progenitor markers using this kit. Mouse cortical stem cells were stained with A. Mouse Anti-SSEA-1 Monoclonal Antibody followed by the NL557-conjugated Anti-Mouse Secondary Antibody (R&D Systems, Catalog # NL019; red), B. Goat Anti-Notch-1 Antigen Affinity-purified Polyclonal Antibody followed by the NL493-conjugated Donkey Anti-Goat Secondary Antibody (R&D Systems, Catalog # NL003; green), and C. Goat Anti-SOX2 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody (R&D Systems, Catalog # NL001). The nuclei were counterstained with DAPI (blue) in each panel. 6 For research use only. Not for use in diagnostic procedures.

9 DATA EXAMPLES CONTINUED A. B. Vimentin/DAPI SOX1/DAPI Figure 3: Verification of Neural Progenitor Marker Expression in Human Neural Stem Cells. Human neural stem cells were assessed for expression of neural progenitor markers using this kit. Human neural stem cells were stained with A. Rat Anti-Vimentin Monoclonal Antibody followed by the NL557-conjugated Goat Anti-Rat Secondary Antibody (R&D Systems, Catalog # NL013; red) and B. Goat Anti-SOX1 Antigen Affinity-purified Polyclonal Antibody followed by the NL557-conjugated Donkey Anti-Goat Secondary Antibody. The nuclei were counterstained with DAPI (blue) in each panel. A. 50 B Relative Cell Number Relative Cell Number CXCR SSEA-1 Figure 4: Detection of Neural Progenitor Markers CXCR4 and SSEA-1 by Flow Cytometry. Cells were stained with antibodies included in this kit. A. CXCR4 was detected in undifferentiated Mouse Cortical Stem Cells (R&D Systems, Catalog # NSC002) using Mouse Anti-CXCR4 Monoclonal Antibody (filled histogram) or Mouse IgG 2A Isotype Control (open histogram). B. SSEA-1 was detected in undifferentiated Rat Cortical Stem Cells (R&D Systems, Catalog # NSC001) using Mouse Anti-SSEA-1 Monoclonal Antibody (filled histogram) or Mouse IgM Isotype Control (empty histogram). Cells were stained using PE-conjugated secondary developing reagents. 7

10 RELATED REAGENTS REAGENT Anti-Nestin Antibody Anti-SOX1 Antibody Anti-SOX2 Antibody Anti-Vimentin Antibody Anti-Notch-1 Antibody Anti-Musashi-1 Antibody Anti-CXCR4 Antibody Anti-SSEA-1 Antibody Goat F(ab') 2 Anti-Mouse IgG (H+L) Allophycocyanin Goat F(ab') 2 Anti-Mouse IgG (H+L) Phycoerythrin Goat F(ab') 2 Anti-Mouse IgG (H+L) Fluorescein Goat F(ab') 2 Anti-Mouse IgG (H+L) PerCP Goat Anti-Mouse IgM Phycoerythrin Goat Anti-Mouse IgM Allophycocyanin Goat Anti-Mouse IgM Fluorescein Goat Anti-Mouse IgM PerCP Flow Cytometry Staining Buffer (1X) Donkey Anti-Goat IgG NL557 Affinity Purified Polyclonal Antibody Donkey Anti-Goat IgG NL637 Affinity Purified Polyclonal Antibody Donkey Anti-Goat IgG NL493 Affinity Purified Polyclonal Antibody Donkey Anti-Mouse IgG NL557 Affinity Purified Polyclonal Antibody Donkey Anti-Mouse IgG NL637 Affinity Purified Polyclonal Antibody Donkey Anti-Mouse IgG NL493 Affinity Purified Polyclonal Antibody Goat Anti-Rat IgG NL557 Affinity Purified Polyclonal Antibody Goat Anti-Rat IgG NL637 Affinity Purified Polyclonal Antibody Goat Anti-Rat IgG NL493 Affinity Purified Polyclonal Antibody Goat Anti-Mouse IgM NL557 Affinity Purified Polyclonal Antibody Goat Anti-Mouse IgM NL493 Affinity Purified Polyclonal Antibody CATALOG NUMBER AF2736, BAF2736, IC1259A, IC1259F, IC1259P, IC2736P, MAB1259, MAB2736, NL1259V, NL2736R AF3369, MAB3369 AF2018, BAF2018, MAB2018, IC2018A, IC2018C, IC2018P, IC2018V, NL20181G, NL2018R AF2105, BAF2105, IC2105P, MAB2105, MAB21051, NL2105R, NL2105V AF1057, AF3647, AF5267, AF5317, BAF1057, BAF3647, BAF5267, BAF5317, BAM53171, FAB5317A, FAB5317P, IC5267C, IC5267P, MAB3647, MAB5267, MAB5317 AF2628, MAB2628 FAB170A, FAB170B, FAB170C, FAB170F, FAB170P, FAB171B, FAB172B, FAB173A, FAB173B, FAB173P, FAB21651A, FAB21651C, FAB21651F, FAB21651P, FABSP2, FABSP2B, MAB170, MAB171, MAB172, MAB173, MAB21651, MAB4287 FAB2155A, FAB2155C, FAB2155P, MAB2155, NL2155R F0101B F0102B F0103B F0114 F0116 F0117 F0118 F0119 FC001 NL001 NL002 NL003 NL007 NL008 NL009 NL013 NL014 NL015 NL019 NL020 8 For research use only. Not for use in diagnostic procedures.

11 REFERENCES 1. McKay, R. (1997) Science 276: Johansson, C.B. et al. (1999) Cell 96: Lendahl, U. et al. (1990) Cell 60: Houle, J. et al. (1983) Dev. Brain Res. 9: MacNicol, A.M. et al. (2008) Biochem. Soc. Trans. 36: Barraud, P. et al. (2005) Eur. J. Neurosci. 22: Ellis, P. et al. (2004) Dev. Neurosci 26: Ni, H.T. et al. (2004) Dev. Brain Res. 152: Imitola, J. et al. (2004) Proc. Natl. Acad. Sci. USA 101: Capela, A. et al. (2002) Neuron 35:

12 NOTES All trademarks and registered trademarks are the property of their respective owners R&D Systems, Inc /14 10 For research use only. Not for use in diagnostic procedures.