Trypsin-EDTA Solution 10X - HiMedia Labs

1 Trypsin-EDTA Solution trypsin and EDTA in normal salineWithout phenol redProduct Code: TCL034 Please refer disclaimer overleafProduct Description :TrypsinMolecular Weight: No: 9002-07-07EC No: is a serine protease derived from porcinepancreas. It is a single chain polypeptide of 223amino acid residue with substrate specificity based onpositively charged Lysine and Arginine side predominantly cleaves peptide chains at thecarboxyl sides of Lysine and Arginine, except wheneither is followed by Proline.

2 It is most commonly usedfor dissociation and disaggregation of adherent acid (EDTA), a chelatingagent is often added to enhance enzymatic activity oftrypsin Solution . EDTA acts by neutralizing calcium andmagnesium ions that enhance cell to cell is trypsin and EDTA in saline. It does not contain phenol :One BAEE unit will produce a r A253nm of perminute with BAEE as substrate at pH at 25 C in areaction volume of (1cm light path).One TAME unit hydrolyzes 1 mole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at25 C, pH , in the presence of calcium USP trypsin unit is the activity causing a changein absorbance of per minute under the Conversion: 1 TAME unit = USP or NFunits = BAEE UnitsDirections :Dilution of 10X Solution to 1X solution1.

3 Thaw frozen trypsin / Trypsin-EDTA Solution in 37 Cwater bath or overnight at 2-8 Aseptically transfer 100ml of 10X trypsin / Trypsin-EDTA Solution to a sterile 1 litre Add 800ml sterile calcium magnesium free balanced saltsolution. Following salt solutions can be used:TL1006 - Dulbecco's Phosphate Buffered SalineTL1034 - Hank's Balanced Salt Solution (With phenol red)TL1098 - Hank's Balanced Salt Solution (Without Phenolred)4. Mix well. Aspirate small quantity of the Solution and takepH. If necessary, adjust the pH to - with sterile 1 NNaOH or 1N Make up the volume to l Filter sterilize the Solution into appropriate of cells from culture vessel1.

4 Remove the spent medium from the culture vessel Wash the monolayer by adding balanced salt solutionwithout calcium and magnesium to the side of the flaskopposite the Rinse the cell sheet by rocking the flask for 1 to 2 minutesand discard the wash Add trypsin or trypsin EDTA Solution to the side ofthe flask opposite the cells. The volume should be sufficientenough to completely cover the monolayer of the Rock the flask to ensure that the dissociation solutioncovers the cell Incubate the flask at 37 C for 2 to 3 minutes.

5 Monitor theprocess by observing the flask under inverted dissociation is complete, the cells will be in suspensionand appear rounded. In addition to rocking gently, flasks ofcell lines that are characteristically difficult to remove fromsubstratum may be tapped to expedite :- The exact time needed to dissociate cells will varyaccording to the cell line. The dissociation process should bemonitored closely to avoid cell Once the cell dissociation is complete add serumcontaining complete medium to the flask to inhibit the trypticactivity which may further damage the Disperse the cells into a single cell suspension by Count and subculture the :User must ensure suitability of the product(s) in their application prior to use.

6 Products conform solely to the information contained in this and otherrelated HiMedia publications. The information contained in this publication is based on our research and development work and is to the bestof our knowledge true and accurate. HiMedia Laboratories Pvt Ltd reserves the right to make changes to specifications and information relatedto the products at any time. Products are not intended for human or animal diagnostic or therapeutic use but for laboratory, research or furthermanufacturing use only, unless otherwise specified.

7 Statements contained herein should not be considered as a warranty of any kind, expressedor implied, and no liability is accepted for infringement of any Laboratories Pvt. Ltd. A-516,Swastik Disha Business Park,Via Vadhani Ind. Est., LBS Marg, Mumbai-400086, India. Customer care No.: 022-6147 1919 Email: Concentration of trypsin or trypsin EDTA solutionused for dissociation should be determined empirically forindividual cell Time required for dissociation of cells from surfacedepends on cell type, cell density, potency of trypsin ,serum concentration in growth medium and time since For serum free media, use Soybean trypsin inhibitor(TCL068) 1:1 to neutralize the action of :-Quality Control.

8 AppearanceColorless, clear bacterial or fungal growth is observed after 14 days ofincubation, as per USP ResponseCell Dissociation ContentNMT 5EU/mlStorage and Shelf Life:Shelf life of the product is 24 receipt store the product at -20 C in a freezer that is notself-defrosting. Once thawed, the product is stable for about2 weeks at 2-8 freezing and thawing reduces enzymatic activityand should be avoided. Once thawed, the Solution can bealiquoted in smaller volumes and frozen for future before expiry date given on the product : 1 / 2012