Description
The BD Stemflow™ Human Neural Lineage Analysis Kit contains a combination of mouse monoclonal antibody conjugates for the analysis of neural differentiation cultures. The antibody specificities that are provided in this kit and the neural cell populations they can identify are listed in the table below. The antibody components of this kit can be used individually or in combinations of up to four. All Antibodies in this kit have been verified by western blot and image analysis. Additional antibody formats that can enable more complex panels are available at www.bdbiosciences.com. The kit also includes BD Cytofix™ Fixation Buffer and BD Phosflow™ Perm Buffer III.
Kit Components
ComponentDescriptionSizeVol. Per TestStorage Buffer
51-9007227PerCP-Cy™5.5 Mouse Anti-Sox225 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007228Alexa Fluor® 647 Mouse Anti-GFAP25 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007229PE Mouse Anti-Doublecortin25 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007230Alexa Fluor® 647 Mouse Anti-Nestin25 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007231Alexa Fluor® 488 Mouse Anti-Human Ki-6725 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007232PerCP-Cy™5.5 Mouse Anti-Human Sox125 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9007233FITC Mouse Anti-Human CD4425 Test5 µlAqueous buffered solution containing
BSA and ≤ 0.09% sodium azide
51-9006607Perm Buffer III50 ml
51-9006276Fixation Buffer50 ml
SpecificityCloneMoleculeNeural cell population identified
CD44G44-26H-CAMGlial cells, astrocytes, astrocyte precursors
Ki-67B56Nuclear proliferation markerAll proliferating cell types
Doublecortin30/DoublecortinNeuroflilamentImmature post-mitotic neurons
Sox2O30-678Transcription factorGlial cells, embryonic and neural stem cells
Sox1N23-844Transcription factorGlial cells and neural stem cells
GFAP1B4FilamentAstrocytesNestin25/NestinFilamentGlial cells, astrocytes, embryonic and neural stem cells
Suggested Companion Products
PerCP-Cy™5.5 Mouse IgG1 κ Isotype Control RUO 0.1mgCat No: 550795
Alexa Fluor® 647 Mouse IgG2b, κ Isotype Control RUO 50TestsCat No: 558713
PE Mouse IgG1, κ Isotype Control RUO 0.1mgCat No: 554680
Alexa Fluor® 647 Mouse IgG1 κ Isotype Control RUO 100TestsCat No: 557732
Alexa Fluor® 488 Mouse IgG1 κ Isotype Control RUO 50TestsCat No: 557782
Perm Buffer III RUO 125mLCat No: 558050
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
Anti-Rat Ig, κ/Negative Control (BSA) Compensation Plus (7.5 µm) Particles Set RUO Cat No: 560499
Stain Buffer (FBS) RUO 500mLCat No: 554656
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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 to Alexa Fluor® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was 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 FITC under optimum conditions, and unreacted FITC was removed.The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was 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.
- Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- 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 PerCP-conjugated product is sold under license to the following patent:US Patent No. 4,876,190.
- Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
(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).Detachment can take up to 45 minutes with mild to moderate triturating of the cell suspension.
(2)Wash the cells in 1X PBS and filter using a 70 μm Falcon® cell strainer (Corning Cat. No. 352350).
(3)Resuspend the cells in BD Cytofix™ Fixation Buffer (Cat. No. 554655) and incubate at room temperature for 20 minutes in the dark.Recommended volumes for fixing cells is 1 ml of BD Cytofix™ Fixation Buffer per 10 million cells with a minimum volume of 200 µl.
(4)Wash the cells twice with 1X PBS. If investigators wish to continue at a later time, cells may be frozen and stored in a solution of 10% DMSO +90% FBS at -80°C using standard methods for the cryopreservation of live cells.
(5)Vortex to loosen the cells and then permeabilize the cells by slowly adding ice cold BD Phosflow™ Perm Buffer III (Cat. No. 558050) while vortexing.Recommended volumes for permeabilizing cells is 1 ml of BD Phosflow™ Perm Buffer III per 10 million cells with a minimum volume of 1 mL.
(6)Incubate on ice for 30 minutes, or alternatively, the cells can be stored in BD Phosflow™ Perm Buffer III for up to 6 months at -20°C.
(7)Wash the cells twice with BD Pharmingen™ Stain Buffer (FBS) (Cat. No.554656) and resuspend at 5 - 10 million cells/ml in BD Pharmingen™ Stain Buffer (FBS) .Alternatively, 1X Washing/Staining Solution (1 x PBS,1% FCS, and 0.09% sodium azide) may be used.
(8)Add 100 µl cells into appropriately labeled 12 x 75mm polystyrene tubes.
(9)Add panel of antibody conjugates that you wish to use ( 5µl/test of each antibody conjugate) and incubate in the dark for 30 minutes.
(10)Wash twice with 2 ml BD Pharmingen™ Stain Buffer (FBS).
(11)Resuspend sample in appropriate volume (350-400µl) ofBD Pharmingen™ Stain Buffer (FBS) to run on a flow cytometer.
Kit Considerations:
Choosing a Cell Detachment Enzyme:Investigators are encouraged to use Accutase™ Cell Detachment Solution (Cat. No. 561527), as cell death with this detachment method has been observed to be minimal. Other methods can be used depending on the confluency and number of cells, such as with 0.05% trypsin.
Fixation and Permeabilization Methods: The components of this kit are all compatible with each other.However if additional specificities are included, those antibody conjugates must be verified to work with the fix/perm buffers included in this kit.
Instrument Set Up:BD™ CompBead Plus particles (Cat. No. 560497 or 560499) may be beneficial in setting up the experiment on a flow cytometer.Investigators are encouraged to set up PMT voltages with the BD™ CompBead Plus particles and then checking to ensure the voltage settings are appropriate by running a stained sample of cells before running the entire experiment.
Data Analysis: A cluster based gating approach is recommended when analyzing data. Backgating to the scatter profile may be useful as the neurons have a distinct scatter population.
In many neural differentiation cultures, it can take a very long time for GFAP to be expressed (greater than 1 month).We have observed that GFAP expression can be augmented by culturing cells in media formulated for astrocyte growth (see below).
Cell debris can sometimes bleed into the neuronal cell population and use of live-dead discriminators can be useful for delineating the neuronal population.
Cell Populations: This kit has been developed for use on neural cell populations that have been derived from human pluripotent stem cells using the methods described in the figure legend below.This kit has not been tested on other cell types, including primary cells, and results may differ when examining cell types of a different origin (e.g higher background staining).In these instances, investigators may find it helpful to use isotype controls to distinguish non-specific background staining from specific antibody staining.
Warnings & Precautions
Danger:BD Phosflow™ Perm Buffer III (component 51-9006607) contains 87.68% methanol (w/w).
Hazard statements
Highly flammable liquid and vapor.
Toxic if swallowed, in contact with skin or if inhaled.
Causes damage to the central nervous system. Route of exposure: Oral.
Precautionary statements
Keep away from heat/sparks/open flames/hot surfaces. - No smoking.
Wear protective clothing / eye protection.
Wear protective gloves.
Do not breathe mist/vapours/spray.
IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.
IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
Danger:Fixation Buffer (component 51-9006276) contains 4.2% formaldehyde (w/w).
Hazard statements
Harmful if inhaled.
Causes skin irritation.
Causes serious eye damage.
May cause an allergic skin reaction.
Suspected of causing genetic defects.
May cause cancer. Route of exposure: Inhalative.
May cause respiratory irritation.
Precautionary statements:
Wear protective clothing / eye protection.
Wear protective gloves.
Do not breathe mist/vapours/spray.
IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.
Continue rinsing.
If skin irritation or rash occurs: Get medical advice/attention.
