University of Pennsylvania
Translational Research Program Laboratory
538-545 BRB 2/3, 421 Curie Boulevard
Philadelphia, PA 19104-6160 / Page 1 of 3
Effective Date:

Document Number: TRP120-01

Title: PHA Stock Preparation

Signature/Approval Date:

Originator:

TRP LabDirector:

1.0 / Purpose
1.1 / This protocol describes the procedures by stock solutions of Phytohemagglutinin (PHA) are prepared and stored.
2.0 / Scope
2.1 / Stock solutions prepared here will be used to stimulate lymphocytes.
3.0 / Definitions and Abbreviations
3.1 / TRP-Translational Research Program
3.2 / ART- Aerosol-Resistant Pipet tips
3.3 / PHA - Phytohemagglutinin
3.4 / PHA-L- (leucoagglutinin), the preferred form with maximal lymphocyte stimulating properties and minimal erythrocyte-agglutinating properties.
3.5 / PHA-P- the whole lectin molecule, with both lymphocyte and erythrocyte-agglutinating properties. This molecule is not preferred.
3.6 / SOP- Standard Operating Procedure
3.7 / PBS- Phosphate Buffered Saline
3.8 / w/o- without
3.9 / Mfr- Manufacturer
3.10 / Cat. - Catalog
3.11 / Exp. - Expiration
4.0 / Responsibility
4.1 / The TRPlaboratory staff is responsible for following this SOP when preparing and storing PHA-L stocks.
4.2 / The TRP laboratory director or his designee will review the records for accuracy.
4.3 / The TRP laboratory staff will archive the records.
5.0 / Equipment and Materials
5.1 / Reagents and Materials (Use listed or equivalent)
5.1.1 / PHA-L from Phaseolus vulgaris, 5 mg, lyophilized (Sigma [800-325-8010] Cat. # L4144).
5.1.2 / ART Pipet tips, various sizes
5.1.3 / #5667 Labels (Office Depot [800-463-3768] Cat. #Item #: 916510)
5.1.4 / PBS w/o Ca2+, Mg2+ (Gibco[800-955-6288], Cat. # 14190-136)
5.1.5 / 15 ml centrifuge tubes (Fisher [1-800-766-7000] Cat. # 14-595-70C)
5.1.6 / Cryotubes, 1.0 ml (Fisher Cat. #12-565-164N)
5.2 / Equipment
5.2.1 / Refrigerator: 2-8oC
5.2.2 / Biosafety Cabinet
5.2.3 / Freezer: -20 +/-5oC
5.3 / Documents
5.3.1 / Form TRP120-A: PHA-L Batch Log
6.0 / Procedures
6.1 / Original powder material should be stored at 2-8oC.
6.1.1 / Product is stable for ~ 6 months under these conditions.
6.2 / Carefully add 1 ml PBS to the vial containing lyophilized reagent.
6.2.1 / Open vial carefully to that lyophilized material does not escape when exposed to atmospheric pressure.
6.3 / Gently swirl and rotate vial to dissolve contents.
6.3.1 / At 5 mg/ml concentration, solution may appear slightly hazy.
6.4 / Transfer 1 ml of dissolved PHA solution to 15 ml conical centrifuge tube.
6.5 / Add 4 ml PBS to 15 ml conical tube, yielding 5 ml of a 1 mg/ml solution.
6.6 / Rinse the vial out using some of the solution to ensure that all the lyophilized powder is transferred.
6.7 / Aliquot by 100 ul into 1.0 ml aliquots.
6.8 / Label the aliquots as follows:
6.8.1 / PHA-L
6.8.2 / 1 mg/ml
6.8.3 / Mfr., Cat. #
6.8.4 / Mfr. Lot #
6.8.5 / Date of Preparation (mm/dd/yy)
6.8.6 / Date of Expiration (6 months from date of preparation or the expiration date on the original vial, whichever comes first)
6.9 / Store aliquots at -20 +/-5oC.
6.10 / Complete form TRP120-A: PHA-L Batch Log
7.0 / Quality Control
7.1 / TRP Lab director or his designee will review form TRP120-A.
7.2 / TRP Lab member will archive form TRP120-A.
8.0 / Special Considerations
8.1 / Universal Precautions will be observed at all times.
8.2 / Aseptic techniques will be used at all times.
8.3 / Thawed aliquots of PHA 1 mg/ml have a shelf life of 1 week at 4oC.
9.0 / References
9.1 / TRP Document D150- Basic Sterile Technique
9.2 / University of Pennsylvania Biological Safety Manual, 2nd edition, 1995
10.0 / Attachments
10.1 / Form TRP120-A PHA Batch Log

11.0Annual Review

Laboratory Director Date

C:\Documents and Settings\carrollr\My Documents\Sops\TI SOPs UPenn CVPF Format\1151-01 Fibronectin Prep Jul 17 02 Draft a.doc