Carrier 5358- User Manual

#5358

Sterile

Human Interleukin-10 (hIL-10)

(Mammalian Derived)

New 07/10

n

SC 10 µg

(With Carrier)

n

LC 50 µg

(With Carrier)

n

SF 10 µg

(Carrier Free)

n

LF 50 µg

(Carrier Free)

Source:

Recombinant human IL-10 (hIL-10) Ser19 -

Asn178 (Accession # NP_000563) was expressed in human
293 cells at Cell Signaling Technology.

Molecular Characterization:

Recombinant hIL-10

contains no “tags” and has a calculated MW of 18,647.
DTT-reduced protein migrates as an 18 kDa polypeptide and
non-reduced protein migrates as a 16 kDa polypeptide due
to intramolecular cystines. The expected amino-terminal
SPGQG of recombinant hIL-10 was verified by amino acid
sequencing.

Endotoxin:

Less than 0.01 ng endotoxin/1

μ

g hIL-10.

Purity:

>98% as determined by SDS-PAGE of 6

μ

g

reduced (+) and non-reduced (-) recombinant hIL-10. All
lots are greater than 98% pure.

Bioactivity:

The bioactivity of recombinant hIL-10 was

determined in a MC/9 cell proliferation assay. The ED

50

of

each lot is between 0.3-1.4 ng/ml.

Formulation:

With carrier: Lyophilized from a 0.22

μ

m filtered

solution of PBS, pH 7.2 containing 20

μ

g BSA per 1

μ

g hIL-10.

Carrier free: Lyophilized from a 0.22

μ

m filtered solution of PBS,

pH 7.2.

Reconstitution:

With carrier: Add sterile PBS, or PBS containing 1% bovine or
human serum albumin or 5-10% FBS to a final hIL-10 concen-
tration of greater than 50

μ

g/ml. Solubilize for 30 minutes at

room temperature with occasional gentle vortexing.

Carrier free: Add sterile PBS, or PBS containing protein to mini-
mize absorption of hIL-10 to surfaces. Solubilize for 30 minutes
at room temperature with occasional gentle vortexing. Stock
hIL-10 should be greater than 50

μ

g/ml.

Storage:

Stable in lyophilized state at 4ºC for 1 year after

receipt. Sterile stock solutions reconstituted with carrier protein
are stable at 4ºC for 2 months and at -20ºC for 6 months. Avoid
repeated freeze-thaw cycles.

Maintain sterility. Storage at -20ºC should be in a manual defrost
freezer.

Applications:

Optimal concentration for the desired application

should be determined by the user.

Background:

IL-10 is an anti-inflammatory cytokine that is pro-

duced by T cells, NK cells and macrophages (1,2). IL-10 initiates
signal transduction by binding to a cell surface receptor complex
consisting of IL-10 RI and IL-10 RII (1). Binding of IL-10 leads
to the activation of Jak1 and Tyk2, which phosphorylates Stat3
(1,3). The anti-inflammatory activity of IL-10 is due to its ability
to block signaling through other cytokine receptors, notably
IFN

γ

receptor, by upregulating expression of SOCS1 (1,3). In

addition, IL-10 promotes T cell tolerance by inhibiting tyrosine
phosphorylation of CD28 (4,5). IL-10 is an important negative
regulator of the immune response, which allows for maintenance
of pregnancy (1). In contrast, increased IL-10 levels contribute to
persistent

Leishmania major

infections (6).

Background References:

(1) Pestka, S. et al. (2004)

Immunol Rev

202, 8-32.

(2) Akuffo, H. et al. (1999)

Clin Exp Immunol

117, 529-34.

(3) O’Shea, J.J. and Murray, P.J. (2008)

Immunity

28, 477-87.

(4) Akdis, C.A. and Blaser, K. (2001)

Immunology

103, 131-6.

(5) Akdis, C.A. et al. (2000)

FASEB J

14, 1666-8.

(6) Von Stebut, E. (2000)

Eur J Dermatol

17, 115-22.

Orders

n

877-616-CELL (2355)
[email protected]

Support

n

877-678-TECH (8324)
[email protected]

Web

n

www.cellsignal.com

Multi-milligram quantities available

© 2010 Cell Signaling T

echnology

, Inc.

This product is intended for research purposes only. This product is not intended to be used for

therapeutic or diagnostic purposes in humans or animals.

The purity of recombinant hIL-10 was determined by SDS-PAGE

of 6 µg reduced (+) and non-reduced (-) recombinant hIL-10

and staining overnight with Coomassie Blue.

kDa

hIL-10

200

116

97
66
55

37

31

22

14

6

4

+

–

The proliferation of MC/9 cells treated with increasing

concentrations of hIL-10 in the presence of 1 pg/ml mouse IL-4

#5208 was assessed. After 72 hours treatment with hIL-10, cells

were incubated with a tetrazolium salt and the OD

450

-OD

650

was

determined.

0

0.4

0.3

0.2

0.1

0.001

0.01

0.1

1

10

100

1000

OD

450

-OD

650

hIL-10 (ng/ml)

Western blot analysis of extracts from RPMI 8226 cells,

untreated or treated with hIL-10 for 10 minutes, using Phospho-

Stat3 (Tyr705) (D3A7) Rabbit mAb #9145 (upper) or Stat3

Antibody #9132 (lower).

kDa

140
100

80

60
50

40
30

20

140
100

80

60
50

40
30

20

0

100

200

10

50

hIL-10 (ng/ml)

Phospho-Stat3

(Tyr705)

Stat3