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For life science research only.

Not for use in diagnostic procedures.

Phytohemagglutinin-L (PHA-L)
From Phaseolus vulgaris
Lyophilizate, before lyophilization filtered through 0.2 ␮m pore size membrane

Cat. No. 11 249 738 001 y Version 15.0


Content version: October 2010
5 mg Store at +2 to +8°C

Product Overview Measuring Growth Stimulation of Peripheral Blood Lympho-


cytes (PBLs)
Formulation Lyophilizate, before lyophilization filtered through
0.2 ␮m pore size membrane. Additional • Peripheral blood Iymphocytes, isolated with
Reagents Iymphocyte separation medium
Preparation Phytohemagglutinin-L (PHA-L) is purified from Phase- Required • Cell culture medium, e.g., RPMI 1640, 10% fetal calf
olus vulgaris (red kidney bean) by standard serum (FCS), 2 mM glutamine, 1% non-essential
chromatographic techniques. amino acids (v/v)
• 1 mg/ml PHA-L dissolved in double dist. sterile
Primary Structure PHA-L is a tetramer of 4 L-type subunits held together water
by noncovalent forces (1). • [3H]-thymidine

Molecular Weight 115,000 Da Procedure


Step Action
Purity PHA-L is ⬎95% pure as determined by SDS-PAGE. 1 900 ␮l cell suspension, 1 × 104 PBLs/ml, are
plated into each well of a microplate.
Biological Optimum activity for the stimulation of proliferation of 2 100 ␮l PHA-L, is added to each well in a dilution
Activity Iymphocytes is at ⬍5 ␮g/ml as tested with human series (stepwidth 1 ␮g, range 1 ␮g–10 ␮g/ml).
peripheral blood Iymphoctes.
3 The mixture is incubated for 48 h at 37°C.
Working For the stimulation of lymphocytes 1–5 ␮g/ml are 4 [3H]-thymidine, 0.5 ␮Ci/well, is added.
Concentration recommended. During flow cytometric analysis, for a 5 The incubation is continued for further 20 h.
maximal shift of the cells into the third cell cycle, PHA-
L ⬍5 ␮g/ml, for a half-maximal shift PHA-L ⬍1 ␮g/ml 6 The cells are collected and washed on glass
is recommended. fiber filter.
7 Remaining cell bound radioactivity is measured
Properties Phytohemagglutinin (PHA), the lectin extract from the in ␣–␤ scintillation counter.
red kidney bean (Phaseolus vulgaris), contains potent,
cell agglutinating and mitogenic activities (1). PHA References 1 Hammerström, S. et al. (1982) Proc. Natl. Acad. Sci. USA 79,
contains a family of five isolectins (L4E0, L3E1, L2E2, L1E3 1611-1616.
, L0E4), each being a tetramer held together by nonco- 2 Leavitt, R.D. et al. (1977) J. Biol. Chem.252, 2961-2966.
valent forces. The subunits are of two different types, 3 Dillner-Centerlind, M. L. et al. (1980) J. Immunol. 10, 434-442.
designated leucocyte reactive (L) and erythrocyte 4 Scott,M.G. & Nahm, M .H. (1984) J. Immunol. 135, 2445-2460.
reactive (E). 5 Di Sabato, G. et al. (1987) Methods Enzymol. 150, 1-17.
6 Parker, C.W. (1987) Methods Enzymol. 150, 29-83.
Type L has high affinity for lymphocyte surface recep-
tors but little for those of erythrocytes and is responsi-
ble for the mitogenic properties of the isolectins. Type E Changes to • Replacement of the wording “sterile filtered”.
is responsible for the erythrocyte agglutinating proper- Previous Version
ties (1, 2). • Editorial changes.
PHA is used for the stimulation of cell proliferation in
lymphocyte cultures (3–6).
PHA-L consists only of L-type subunits (isolectin L4,
“leukoagglutinin”) and is especially suitable for high
efficiency induction of T-lymphocytes.
Application PHA-L should be used as a high quality tool for func-
tional analysis of T-lymphocytes. It is highly purified
and tested for high efficiency stimulation of human
lymphocytes.
Note: Although PHA-L may be employed for routine Contact and Support
lymphocyte culture, the conditions necessary for its
successful use are much more stringent then those To ask questions, solve problems, suggest enhancements or report new
required for less pure reagents. In particular, the con- applications, please visit our Online Technical Support Site at:
centration of PHA-L giving maximal lymphocyte stimu-
lation without inhibition or toxicity lies within very www.roche-applied-science.com/support
narrow limits.
To call, write, fax, or email us, visit the Roche Applied Science home page,
Reconstitution PHA-L should be reconstituted in sterile water (final www.roche-applied-science.com, and select your home country. Country-
concentration: 1 mg/ml). Further dilution with medium specific contact information will be displayed. Use the Product Search func-
or PBS (phosphate buffered saline). tion to find Pack Inserts and Material Safety Data Sheets.

Stability The Iyophilizate is stable at +2 to +8°C until the expira-


tion date printed on the label. The reconstituted solu-
tion is stable at ⫺15 to ⫺25°C.
Note: It is recommended to store the reconsituted Roche Diagnostics GmbH
solution in aliquots at ⫺15 to ⫺25°C, to avoid repeated Roche Applied Science
freezing and thawing. 68298 Mannheim
Germany
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