AP Staining Kit User Manual
Contents
1. Introduction and Background A Overview Alkaline phosphatase AP is a universal pluripotent marker for all types of pluripotent stem cells including embryonic stem cells embryonic germ cells and induced pluripotent stem cells The pluripotent status of stem cells can be characterized by a high level of AP expression along with the expression of multiple pluripotency markers including the transcription factors Nanog Oct4 Sox2 stage specific embryonic antigens SSEA 1 3 4 and tumor related antigens TRA 1 60 TRA 1 81 Cell type APs SSEA 1 SSEA4 TRA 1 60 TRA 1 81 Mouse ES Cell X z Mouse EG Cell 4 Human ES Cell Kj Human EG Cell Ki Kj Human EC Cell X x ES cell Embryonic stem cell EG cell Embryonic germ cell EC cell Embryonic carcinoma cell The Dual Color AP staining kit is a specific sensitive and reliable tool for phenotypic assessment of AP expression at the surface of stem cells The Dual Color AP kit provides sufficient reagents for a total of 100 assays 24 well plate formate with both Red and Blue 50 assays for each color The AP Red 50 assays or AP Blue kits 50 assays are also sold separately Alkaline phosphatase AP is a hydrolase enzyme responsible for dephosphorylating molecules such as nucleotides and proteins under alkaline conditions The Dual Color Alkaline Phosphatase Staining Kit is a histochemical assay for use on cells grown in tissue culture wells or dishes It uses a sol2. Cat AP100 D 1 Fixation Solution 25 ml X 2 Solution A Blue 10 ml Solution B Blue 10 ml Solution A Red 10 ml Solution B Red 10 ml E Materials Needed but Not Supplied e Human mouse embryonic stem cells or induced pluripotent stem cells e 1X PBS e Light Microscope F Storage Store all components at 4 C until their expiration dates Page 4 ver 1 080610 www systembio com AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 ll AP Staining Protocol A Preparation of Reagents Prepare sufficient AP substrate solution for each experiment Quantities can be scaled up using a 1 1 ratio by volume of Solution A and Solution B For optimal results the AP substrate solution should be used within 30 minutes of preparation The AP substrate solution can remain at room temperature afterwards B Alkaline Phosphatase Staining 1 Culture mouse human stem cells on feeder layer or feeder free 2 Gently aspirate the culture medium and wash the cells with 1 ml of 1X PBS Aspirate the wash solution 3 Add Fix Solution to the cells 0 5 ml per well for a 24 well plate Incubate at room temperature for 2 to 5 min Do not fix your cells beyond 20 min as over fixation may result in the loss of AP activity 4 Aspirate the Fix Solution and wash the fixed cells with 1X PBS twice 5 Remove the 1X PBS and add freshly prepared AP Subsirate Solution into each well For a 24 well plate add 0 4 ml per well 6 Incubate the cells
3. with substrate at room temperature for 15 to 20 minutes protected from light 7 Stop the reaction by aspirating the staining Solution and rinsing the wells twice with 1X PBS 8 Cover the cells with 1X PBS to prevent drying out 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 9 AP expression will result in a red purple stain for Color Red and a blue stain for Color Blue while the absence of AP expression will result in no stain 10 Observe the red blue stained cell colonies undifferentiated stem cells vs colorless colonies differentiated stem cells or non stem cells using a light microscope Store the plate at 4 C for future analysis up to one week C Example of Results The following figures demonstrate typical results with the Dual Color AP staining kit Staining of mouse induced pluripotent stem cells using the Blue Color and Red Color AP staining kit AP positive colonies appear either blue or red to purple indicated by blue or red arrow while the surrounding feeder cells appear colorless Blue Color Kit Red Color Kit Page 6 ver 1 080610 www systembio com AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 Use the AP staining kit to discriminate between pluripotent versus differentiated cells as shown below Staining of mouse induced pluripotent stem cells using the Red Color AP staining kit The differentiated colony indicated by white arrow appear
4. 2 SBI ystem Biosciences AP Staining Kit Cat AP100B 1 Cat AP100R 1 Cat AP100D 1 User Manual Store 4 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement ver 1 101910 contained in this user manual AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 Contents I Introduction and Background ceeeeeeeeeneeeeeeenees As OV GINIO Weiss cucaticuats bislent paheseecthasounssdianeae Sees B Suggested Usage cccceceesececeeceeeeeeeesseeeeneeeeneees C Workflow of AP Staining ccccceeeeeseeeeeseeeeeeees D Kit COMponentS c cceeeeeeeeeeeeeeeeeeeeeaeeeeaeeeeneeees E Materials Needed but Not Supplied e Fe Storage ies a a A seiutew cheat EA Il AP Staining Protocol seesseesseesseessreesrnesrnesrnssrnssrnssns A Preparation of Reagent ccceesseeeeeesieeeeeeeieeeeeeee B Alkaline Phosphatase Staining cccsseeeseeeeerees C Example of ReSUuIts c cccccceeseeeeeeeeeeeeeseeeeeeneeeeenees III Related Products asssessssssrresssrrsserrresrinnesrennesrnnnes IV References s c00 i eie a i V Technical Support ssssssesseseesresrssrssresrresrresrresres VI Licensing and Warranty Statement 0 ce 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual
5. e actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2010 System Biosciences SBI All Rights Reserved 888 266 5066 Toll Free 650 968 2200 outside US Page 11
6. s colorless and undifferentiated colony indicated by red arrow appears red or purple 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual AP Staining Picture Grid Fixation Time Blue Color Red Color 2 min 5 min 10 min 2 min 5 min 10 min 5 min 10 min 20 min 30 min lll Related Products Pluripotency reporters http systembio com stem cell research pluripotency reporters catalog listings Differentiation reporters http systembio com stem cell research differentiation reporters catalog listings High quality feeder cells and source cells and Human Mouse iPS cell lines http systembio com viral ipscs viral ipscs tab 1 5 Page 8 ver 1 080610 www systembio com AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 References 1 Takahashi K Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126 4 663 76 2 Takahashi K Tanabe K Ohnuki M Narita M Ichisaka T Tomoda K Yamanaka S 2007 Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Cell 131 861 872 3 YudJ Vodyanik M A Smuga Otto K Antosiewicz Bourget J Frane J L Tian S Nie J Jonsdottir G A Ruotti V Stewart R Slukvin l l Thomson J A 2007 Induced pluripotent stem cell lines deri
7. to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Page 10 ver 1 080610 www systembio com AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to th
8. ution containing substrate and color dye as the colorimetric read out for the enzymatic activity The AP positive undifferentiated stem cells Page 2 ver 1 080610 www systembio com AP Staining Kit Cat AP100B 1 AP100D 1 AP100R 1 stain red or blue The stain develops quickly does not readily fade and can be analyzed 15 to 30 minutes after application to the cultured cells For added convenience the stained samples can be kept at 4 C for up to one week for analysis B Suggested Usage e Measure alkaline phosphatase activities in situ Immunohistochemistry e Quality control of stem cells e Monitor cell differentiation pluripotency status C Workflow of AP Staining Step 1 Seed stem cells in culture Day 1 Step 2 Perform experiment Day 3 5 Step 3 AP Staining total time 2 hours 1 Prepare Staining Solution 2 Wash twice with 1X PBS 3 Fix cells for 2 5 minutes 4 Wash twice with 1X PBS 5 Incubate for 15 20 min in the dark at room temperature 6 Stop reaction by washing twice with 1X PBS KP 7 Data analysis mean 2 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual D Kit Components Blue Color AP Staining Kit Cat AP100 B 1 Fixation Solution 25 ml Solution A Blue 10 ml Solution B Blue 10 ml Red Color AP Staining Kit Cat AP100 R 1 Fixation Solution 25 ml Solution A Red 10 ml Solution B Red 10 ml Dual Color AP Staining Kit
9. ved from human somatic cells Science 318 1917 1920 Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 1616 North Shoreline Blvd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 9 Vi System Biosciences SBI User Manual Licensing and Warranty Statement Limited Use License Use of the Dual Color AP Staining Kit i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used
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