BD, 562271, BD Stemflow™ Human Neural Cell Sorting Kit

Reactivity: Human (Reactivity Confirmed in Development)
Regulatory Status: RUO
RRID: AB_2869412
Description: 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: 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.
Recommended Assay Procedures: 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