BD, 562496, BD Stemflow™ Human Definitive and Pancreatic Endoderm Analysis Kit

Reactivity: Human (QC Testing)
Application: Intracellular staining (flow cytometry) (Routinely Tested)
Regulatory Status: RUO
RRID: AB_2869421
Description: Description The BD Stemflow™ Human Definitive and Pancreatic Endoderm Analysis Kit contains a combination of mouse monoclonal antibody conjugates for the analysis of definitive and pancreatic endodermal differentiation cultures. The antibody specificities that are provided in this kit and the 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.  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 51-9008006 Alexa Fluor® 488 Mouse Anti-Human Pax-6 25 Test 5 µl Aqueous buffered solution containing BSA and ≤0.09% sodium azide 51-9008007 PE Mouse Anti-PDX-1 25 Test 5 µl Aqueous buffered solution containing BSA and ≤0.09% sodium azide 51-9008008 PE Mouse Anti-Human FoxA2 25 Test 5 µl Aqueous buffered solution containing BSA and ≤0.09% sodium azide 51-9007227 PerCP-Cy™5.5 Mouse Anti-Sox2 25 Test 5 µl Aqueous buffered solution containing BSA and ≤0.09% sodium azide 51-9008009 PerCP-Cy™5.5 Mouse Anti-Human Sox17 25 Test 5 µl Aqueous buffered solution containing BSA and ≤0.09% sodium azide 51-9008010 Alexa Fluor® 647 Mouse Anti-Human Nanog 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 Cell Population Identified Nanog N31-335 Pluripotent stem cells Sox2 O30-678 Pluripotent stem cells, ectodermal contaminants Sox17 P7-969 Definite endoderm FoxA2 N17-280 Definitive endoderm Pax6 O30-1330 Pancreatic endoderm, ectodermal contaminants Pdx1 658A5 Pancreatic endoderm
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). Mild to moderate triturating of the cell suspension is recommended to achieve a single cell suspension. 2. Wash the cells in 1X PBS. If clumps are present, cells can be filtered using a 70 mm Falcon® cell strainer (Corning Cat. No. 352350). 3. Resuspend the cells in 0.5X BD Cytofix™ Fixation Buffer (Cat. No. 554655) diluted in 1X PBS and incubate at 37°C for 10 minutes. Recommended volumes for fixing cells is 1 ml of fixation solution 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 cell pellet 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 (5 μL/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 (350-400 μL) 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. 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. Addition of Cell Surface Markers: CD184 PE (Cat. No. 555974), CD184 APC (Cat. No. 555976), and CD184 BV421 (Cat. No. 562448 ) have been used successfully with components of this kit. However, as non-specific background staining is seen on pluripotent stem cells that have been fixed then stained with CD184, it is recommended that cells are detached, stained live with CD184, fixed as recommended above, permeabilized with 0.1% Triton™ X-100 (Perm III can negatively impact CD184 positive stain), and then stained for intracellular markers. Instrument Set Up: BD™ CompBead Plus particles (Cat. No. 560497) 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. Cell Populations: This kit has been developed for use on endodermal cell populations that have been derived from human pluripotent stem cells using the methods described in the figure legends 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: BD Cytofix™ 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.