BD, 561526, BD Stemflow™ Human Neural Lineage Analysis Kit

Reactivity: Human (QC Testing)
Application: Flow cytometry (Routinely Tested)
Regulatory Status: RUO
RRID: AB_2869383
Description: 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 Component Description Size Vol. Per Test Storage Buffer 51-9007227 PerCP-Cy™5.5 Mouse Anti-Sox2 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007228 Alexa Fluor® 647 Mouse Anti-GFAP 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007229 PE Mouse Anti-Doublecortin 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007230 Alexa Fluor® 647 Mouse Anti-Nestin 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007231 Alexa Fluor® 488 Mouse Anti-Human Ki-67 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007232 PerCP-Cy™5.5 Mouse Anti-Human Sox1 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9007233 FITC Mouse Anti-Human CD44 25 Test 5 µl Aqueous buffered solution containing BSA and ≤ 0.09% sodium azide 51-9006607 Perm Buffer III 50 ml 51-9006276 Fixation Buffer 50 ml Specificity Clone Molecule Neural cell population identified CD44 G44-26 H-CAM Glial cells, astrocytes, astrocyte precursors Ki-67 B56 Nuclear proliferation marker All proliferating cell types Doublecortin 30/Doublecortin Neuroflilament Immature post-mitotic neurons Sox2 O30-678 Transcription factor Glial cells, embryonic and neural stem cells Sox1 N23-844 Transcription factor Glial cells and neural stem cells GFAP 1B4 Filament Astrocytes Nestin 25/Nestin Filament Glial cells, astrocytes, embryonic and neural stem cells
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 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.
Recommended Assay Procedures: Recommended Assay Procedures (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 μ m 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) of  BD 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.