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
ComponentDescriptionSizeVol. Per TestStorage Buffer
51-9007757PE Mouse Anti-Human CD2420 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007758PerCP-Cy™5.5 Mouse Anti-Human CD27120 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007759PerCP-Cy™5.5 Mouse Anti-Human CD4420 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007760PE-Cy™7 Mouse Anti-Human CD1520 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007761APC Mouse Anti-Human CD18420 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007762PE Mouse IgG1, κ Isotype Control10 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007763PerCP-Cy™5.5 Mouse IgG1, κ 10 Test5 µlAqueous buffered solution containing
Isotype Control for 51-007758BSA and <0.09% sodium azide
51-9007764PerCP-Cy™5.5 Mouse IgG1, κ 10 Test5 µlAqueous buffered solution containing
Isotype Control for 51-9007759BSA and <0.09% sodium azide
51-9007765PE-Cy™7 Mouse IgM, κ Isotype Control10 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007766APC Mouse IgG1, κ Isotype Control10 Test5 µlAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007767Negative Control CompBead Plus1.5 ml1 dropAqueous buffered solution containing
BSA and <0.09% sodium azide
51-9007768Anti-Mouse Ig, κ CompBead Plus1.5 ml1 dropAqueous buffered solution containing
BSA and <0.09% sodium azide
SpecificityCloneMoleculeNSC PhenotypeNeuron PhenotypeGlial Phenotype
CD15HI98X-hapten, SSEA-1-/+ LowNA
CD24ML5Heat Stable Antigen + +NA
CD44G44-26H-CAM- -+
CD18412G5CXCR4, Fusin + -+
CD271C40-1457NGF-Receptor- NANA
Suggested Companion Products
Accutase™ Cell Detachment Solution RUO 100mLCat No: 561527
Anti-Mouse Ig, κ/Negative Control (BSA) Compensation Plus (7.5 µm) Particles Set RUO Cat No: 560497
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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.
Product Notices
- 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.
- 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.
- 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.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
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 labelAdd (1 test/drop)
1. Unlabeled compensationNegative CompBead plus + Anti-mouse Compbead plus
2. PE compensationNegative CompBead plus + Anti-mouse Compbead plus + CD24 PE
3. PerCP-Cy™5.5 compenstionNegative CompBead plus + Anti-mouse Compbead plus + CD271 PerCP-Cy™5.5
4. PE-Cy7 compensationNegative CompBead plus + Anti-mouse Compbead plus + CD15 PE Cy™7
5. APC CompensationNegative CompBead plus + Anti-mouse Compbead plus + CD184 APC
6. Cells aloneNothing
7. Isotype controlMs 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 sampleCD24 PE + CD44 PerCP-Cy5.5 + CD271 PerCP-Cy5.5 + CD15 PE-Cy7 + CD184 APC
Neuron and Glia Sort
Tube labelAdd (1 test/drop)
1. Unlabeled compensationNegative CompBead plus + Anti-mouse Compbead plus
2. PE compensationNegative CompBead plus + Anti-mouse Compbead plus + CD24 PE
3. PerCP-Cy™5.5 compenstionNegative CompBead plus + Anti-mouse Compbead plus + CD44 PerCP-Cy™5.5
4. PE-Cy7 compensationNegative CompBead plus + Anti-mouse Compbead plus + CD15 PE Cy™7
5. APC CompensationNegative CompBead plus + Anti-mouse Compbead plus + CD184 APC
6. Cells aloneNothing
7. Isotype controlMs IgG1, k PE + Ms IgG1 PerCP-Cy5.5 (CD44 ITCL) + Ms IgM PE-Cy7 + Ms IgG1 APC
8. Sort sampleCD24 PE + CD44 PerCP-Cy5.5 + CD15 PE-Cy7 + CD184 APC
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