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      Human Neural Cell Sorting Kit

      BD Stemflow™ Human Neural Cell Sorting Kit

      Human Neural Cell Sorting Kit
      Figure 1  Cell sorting of NSC from neural induction cultures: H9 hESC were differentiated into neural ectoderm using SFEB and dual SMAD inhibition.  Please see Recommended Assay Procedure for more details.  On day 20 of differentiation cells were processed as described above and sorted using the following gating strategy. Neural Stem Cell Sort (Gates based upon isotype controls): 1.        Identify cells in FSC v SSC plot. (P1) a.        Doublet discrimination should be applied after this step (P2) 2.        Create a child gate from P2 that includes the CD184+ cells (P3). 3.        Create a child gate from P3 that includes CD44- and CD271- cells (P4)           4.        Create a child gate from P4 that in cludes the CD24+CD15+/- (NSC) cells Figure 2  Cell sorting of neurons and glia from differentiating NSC cultures: Previously sorted and expanded NSC derived from H9 hESC were differentiated for ~ 2.5 weeks in DMEM:F12+Glutamax, 1X B27, 1X N2 (Invitrogen), 1X P/S (Lonza), 20 ng/ml BDNF, 20ng/ml GDNF (both from Peprotech) and 0.5 mM dibutyryl cyclic AMP (Sigma).  Cells were processed as described above and sorted using the following gating strategy. Neuron and Glia Sort (Gates based upon isotype controls): 1.        Identify cells in FSC v SSC plot. (P1) a.        Doublet discrimination should be applied after this step. (P2) b.        Make sure to include the glia as they appear as a distinct scatter population in more mature cultures. 2.        Create a child gates from P2 that includes CD44-CD184- cells (P3) or CD44+CD184+ cells (glia)           3.        Create a child gate from P3 that includes the CD24+CD15- cells (Neurons)
      Human Neural Cell Sorting Kit
      Figure 1  Cell sorting of NSC from neural induction cultures: H9 hESC were differentiated into neural ectoderm using SFEB and dual SMAD inhibition.  Please see Recommended Assay Procedure for more details.  On day 20 of differentiation cells were processed as described above and sorted using the following gating strategy. Neural Stem Cell Sort (Gates based upon isotype controls): 1.        Identify cells in FSC v SSC plot. (P1) a.        Doublet discrimination should be applied after this step (P2) 2.        Create a child gate from P2 that includes the CD184+ cells (P3). 3.        Create a child gate from P3 that includes CD44- and CD271- cells (P4)           4.        Create a child gate from P4 that in cludes the CD24+CD15+/- (NSC) cells Figure 2  Cell sorting of neurons and glia from differentiating NSC cultures: Previously sorted and expanded NSC derived from H9 hESC were differentiated for ~ 2.5 weeks in DMEM:F12+Glutamax, 1X B27, 1X N2 (Invitrogen), 1X P/S (Lonza), 20 ng/ml BDNF, 20ng/ml GDNF (both from Peprotech) and 0.5 mM dibutyryl cyclic AMP (Sigma).  Cells were processed as described above and sorted using the following gating strategy. Neuron and Glia Sort (Gates based upon isotype controls): 1.        Identify cells in FSC v SSC plot. (P1) a.        Doublet discrimination should be applied after this step. (P2) b.        Make sure to include the glia as they appear as a distinct scatter population in more mature cultures. 2.        Create a child gates from P2 that includes CD44-CD184- cells (P3) or CD44+CD184+ cells (glia)           3.        Create a child gate from P3 that includes the CD24+CD15- cells (Neurons)
      Product Details
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      BD Stemflow™
      Human (Reactivity Confirmed in Development)
      RUO
      AB_2869412


      Description

      The BD Stemflow™ Neural Cell Isolation Kit was designed to allow the isolation of neural stem cells (NSCs) derived from human pluripotent stem cells or the isolation of neurons and glial cells from differentiated NSCs.  Following a variety of established neural induction protocols, NSCs are sorted from the heterogeneous cell culture utilizing the included antibody conjugates to five cell surface markers.  This method greatly reduces the amount of variability between NSC isolations as opposed to traditional methods like manual isolation.  In addition, four of the five included conjugates can be used to isolate neurons from glia from differentiated NSCs.

      Kit Components

      Component        Description                                                                        Size                Vol. Per Test        Storage Buffer

      51-9007757        PE Mouse Anti-Human CD24                                        20 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007758        PerCP-Cy™5.5 Mouse Anti-Human CD271                        20 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007759        PerCP-Cy™5.5 Mouse Anti-Human CD44                        20 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007760        PE-Cy™7 Mouse Anti-Human CD15                                20 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007761        APC Mouse Anti-Human CD184                                        20 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007762        PE Mouse IgG1, κ Isotype Control                                10 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007763        PerCP-Cy™5.5 Mouse IgG1, κ                                        10 Test                5 µl                Aqueous buffered solution containing

                              Isotype Control for 51-007758                                                                                BSA and <0.09% sodium azide

      51-9007764        PerCP-Cy™5.5 Mouse IgG1, κ                                        10 Test                5 µl                Aqueous buffered solution containing

                              Isotype Control for 51-9007759                                                                                BSA and <0.09% sodium azide

      51-9007765        PE-Cy™7 Mouse IgM, κ Isotype Control                        10 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007766        APC Mouse IgG1, κ Isotype Control                                10 Test                5 µl                Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007767        Negative Control CompBead Plus                                        1.5 ml                1 drop        Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      51-9007768        Anti-Mouse Ig, κ CompBead Plus                                        1.5 ml                1 drop        Aqueous buffered solution containing

                                                                                                                                                              BSA and <0.09% sodium azide

      Specificity        Clone                Molecule                        NSC Phenotype                Neuron Phenotype                Glial Phenotype

      CD15                HI98                X-hapten, SSEA-1                    -/+                                 Low                                NA

      CD24                ML5                Heat Stable Antigen             +                                         +                                        NA

      CD44                G44-26                H-CAM                                      -                                         -                                        +

      CD184                12G5                CXCR4, Fusin                             +                                         -                                        +

      CD271                C40-1457        NGF-Receptor                              -                                         NA                                        NA

      Preparation And Storage

      Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. The antibody was conjugated with PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity. The antibody was conjugated with PE-Cy7 under optimum conditions, and unconjugated antibody and free PE-Cy7 were removed. The antibody was conjugated to APC under optimum conditions, and unconjugated antibody and free APC were removed.
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      Recommended Assay Procedures

      We have had consistent results with the H9 (WiCell, Madison WI) human embryonic stem cell (hESC) line and have followed established serum free embryoid body neural induction methods in combination with dual SMAD inhibition (Noggin and SB431542).  For neuronal differentiation we recommend differentiating NSC for at least 3 weeks as the CD44/CD184 double positive glial population requires longer culturing times to differentiate.  (Please see Yuan et al. PLoS One. 2011 Mar 2;6(3):e17540 for details).  As cells and culturing methods can differ, we recommend a time course analysis to determine appropriate timing for cell sorting experiments.

              

      (All steps performed using sterile techniques)

      1.        Detach cells of interest from the culture dish. Investigators are encouraged to detach cells at 37°C using Accutase™ Cell Detachment Solution (Cat. No. 561527).

      a.        If needed some mild trituration may be used to better obtain a single cell suspension.

      b.        Neuron disassociation may take up to 45 minutes.

      2.        Collect and spin down cells.  Resuspend cells in 10ml of basal w/supplements (e.g. DMEM/F12 + 1X N2 + 1XB27) media containing 100 u/ml DNAse.  Incubate at room temperature for 10 minutes.

      3.        Filter cells through a 70 µm cell strainer and then spin down cells and resuspend cells at 10 million cells/ml in basal media w/supplements and also with 5 mM EDTA + 0.5 percent BSA.

      4.        Label tubes and add the corresponding antibody conjugates as shown (use appropriate tables listed below for respective cell prep)

      a.        Use sterile 12x75mm tubes with caps.

      5.        Add 100 µl of cells to tubes 6 and 7

      6.        Add up to 1 ml of cells to tube 8.

      a.        If you wish to sort more than 10 million cells we recommend replicating tube 8 with additional cells that you wish to sort.

      7.        Incubate cells on ice in the dark for 20-30 minutes.

      8.        Wash once with 2 ml basal media w/supplements and also 5 mM EDTA + 0.5 percent BSA.

      9.        Resuspend cells in basal media w/supplements and also 5 mM EDTA + 0.5 percent BSA at a concentration of 2.5 to 5 million cells/ml

      a.        Alternatively please contact your cell sorter operator to get a suggested final concentration of cells for sorting.

                              Neural Induction Staining (NSC sort)

              Tube label                                        Add (1 test/drop)

              1. Unlabeled compensation                Negative CompBead plus + Anti-mouse Compbead plus

              2. PE compensation                        Negative CompBead plus + Anti-mouse Compbead plus + CD24 PE

              3. PerCP-Cy™5.5 compenstion        Negative CompBead plus + Anti-mouse Compbead plus + CD271 PerCP-Cy™5.5

              4. PE-Cy7 compensation                Negative CompBead plus + Anti-mouse Compbead plus + CD15 PE Cy™7

              5. APC Compensation                        Negative CompBead plus + Anti-mouse Compbead plus + CD184 APC

              6. Cells alone                                        Nothing

              7. Isotype control                                Ms IgG1, k PE +  Ms IgG1 PerCP-Cy5.5 (CD44 Isotype Control) + Ms IgG1 PerCP-Cy5.5 (CD271                                                                         Isotype Control) +         Ms IgM PE-Cy7 +  Ms IgG1 APC

              8. Sort sample                                        CD24 PE + CD44 PerCP-Cy5.5 + CD271 PerCP-Cy5.5 + CD15 PE-Cy7 + CD184 APC

                              Neuron and Glia Sort

              Tube label                                        Add (1 test/drop)

              1. Unlabeled compensation                Negative CompBead plus + Anti-mouse Compbead plus

              2. PE compensation                        Negative CompBead plus + Anti-mouse Compbead plus + CD24 PE

              3. PerCP-Cy™5.5 compenstion        Negative CompBead plus + Anti-mouse Compbead plus + CD44 PerCP-Cy™5.5

              4. PE-Cy7 compensation                Negative CompBead plus + Anti-mouse Compbead plus + CD15 PE Cy™7

              5. APC Compensation                        Negative CompBead plus + Anti-mouse Compbead plus + CD184 APC

              6. Cells alone                                        Nothing

              7. Isotype control                                Ms IgG1, k PE + Ms IgG1 PerCP-Cy5.5 (CD44 ITCL) + Ms IgM PE-Cy7 + Ms IgG1 APC

              8. Sort sample                                        CD24 PE + CD44 PerCP-Cy5.5 + CD15 PE-Cy7 + CD184 APC

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      Product Notices

      1. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
      2. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
      3. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
      4. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
      5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
      6. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
      7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      562271 Rev. 2
      Citations & References
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      Development References (2)

      1. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009; 27(3):275-280. (Biology: Cell differentiation). View Reference
      2. Yuan SH, Martin J, Elia J, et al. Cell-Surface Marker Signatures for the Isolation of Neural Stem Cells, Glia and Neurons Derived from Human Pluripotent Stem Cells. PLoS ONE. 6(3)(Methodology: Flow cytometry). View Reference
      562271 Rev. 2

      Please refer to Support Documents for Quality Certificates

      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

      Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

      For Research Use Only. Not for use in diagnostic or therapeutic procedures.