Mouse Interleukin-7 Recombinant

$70.00 – $4,700.00

SKU: RKP10168 Tags: il-7, il7, Interleukin-7, P10168

Description

Accession

P10168

Source

Optimized DNA sequence encoding Human Interleukin-7 mature chain was expressed in Escherichia Coli.

Molecular weight

Native Mouse IL-7, generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa. Recombinant mouse IL-7 is a monomeric protein consisting of amino acid residue subunits. Recombinant IL-7 migrates as an approximately kDa protein under non-reducing and reducing conditions in SDS-PAGE.

Purity

>95%, as determined by SDS-PAGE and HPLC

Biological Activity

The ED(50) dose-dependent stimulation of the proliferation of murineE8 cells is <.1 ng/ml, corresponding to a specific activity of >4 x units/mg.

Protein Sequence

MFHVSFRYIF GIPPLILVLL PVTSSECHIK DKEGKAYESV LMISIDELDK MTGTDSNCPN NEPNFFRKHV CDDTKEAAFL NRAARKLKQF LKMNISEEFN VHLLTVSQGT QTLVNCTSKE EKNVKEQKKN DACFLKRLLR EIKTCWNKIL KGSI

Endotoxin

Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than 0.1 ng/µg(1EU/µg).

Presentation

Recombinant Interleukin-7 was lyophilized from a 0.2 μm filtered solution in.5% glycine,.5% sucrose,.01% Tween80, mM Glutamic acid, pH.5.

Reconstitution

A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 mg/mL. This solution can then be diluted into other buffers.

Storage

The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at2° -8° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.

Usage

This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Molecular function

Cytokine

Molecular function

Growth-factor

Methods

T cell activation assay

Stromal cells:24-well 1×10⁴ stromal cells from lines were seeded in complete RPMI and after overnight culture irradiated with 1000rad.
Initially, experiments with non-irradiated or irradiated pLN2 were performed with no difference in the outcome (data not shown).
Ex vivo stromal cells were isolated as described above and non-adherent cells washed away after overnight culture (density comparable with lines); they were non-proliferative and therefore not irradiated.
BMDC: They were activated with 0.5 µg/ml LPS for 6 h at 37°C.
2 h after LPS addition 1 µM SIINFEKL peptide was added.
1×10⁴ BM-DC were added per 24-well.
T cells: They were obtained ex vivo from spleen and pLN dissected from CO₂-killed WT B6 and OT-I transgenic mice and suspended by meshing.
Erythrocytes were removed using red blood cell lysis buffer (Tris-Ammonium chloride based).
CD8 cells were enriched by panning using antibodies to B220 (RA3-6B2), CD4 (H129.19.6), CD11b (M1/70) and CD11c (N418).
OT-I cells were labeled…

Stromal cells:24-well 1×10⁴ stromal cells from lines were seeded in complete RPMI and after overnight culture irradiated with 1000rad.
Initially, experiments with non-irradiated or irradiated pLN2 were performed with no difference in the outcome (data not shown).
Ex vivo stromal cells were isolated as described above and non-adherent cells washed away after overnight culture (density comparable with lines); they were non-proliferative and therefore not irradiated.
BMDC: They were activated with 0.5 µg/ml LPS for 6 h at 37°C.
2 h after LPS addition 1 µM SIINFEKL peptide was added.
1×10⁴ BM-DC were added per 24-well.
T cells: They were obtained ex vivo from spleen and pLN dissected from CO₂-killed WT B6 and OT-I transgenic mice and suspended by meshing.
Erythrocytes were removed using red blood cell lysis buffer (Tris-Ammonium chloride based).
CD8 cells were enriched by panning using antibodies to B220 (RA3-6B2), CD4 (H129.19.6), CD11b (M1/70) and CD11c (N418).
OT-I cells were labeled with 2 µM CFSE .
Only 50% of WT B6 T cells were CFSE-labeled to identify the peak of undivided cells, with the 50% unlabeled T cells showing the background fluorescence of T cells.
Per 24-well 0.04×10⁶ CFSE⁺ OT-I cells together with 0.98×10⁶ unlabeled B6 WT cells and 0.98×10⁶ CFSE⁺ B6 WT cells were added.
The assay was performed in complete RPMI enriched with 1×MEM , 3 ng/ml murine IL-7 and 10 U/ml human IL-2 .
After 2–4 days, cells were harvested, counted and analyzed by flow cytometry.
Transwell assays used were 0.4 µm transwell chambers (HTS 24-well).
Blocking experiments used 10 µM indomethacin , 1 µM 1400W ( = dihydrochloride), 10 µM 1-Methyl-L-tryptophan (1-MT), 200 µM (S)-(2-Boronoethyl)-L-cysteine , 10 µg/ml anti-PD-L1 (MIH5, eBioscience), 20 µg/ml anti-IL-10 (kind gift from, , ) or 30 µg/ml anti-TGFβ (clone 1D11.16.8).
To better compare the decrease of T cell proliferation between experiments the attenuating effect by the stromal cells was defined as percent inhibition in divided OT-I T cell numbers relative to the ‘no stroma’ control: {1-(number of proliferated cells)_stroma/(number of proliferated cells)_{no stroma}}*100).
The ‘no stroma’ control was considered as 0% inhibition; absence of proliferating OT-I cells was considered as 100% inhibition.

Bone marrow was harvested from C57BL/6 mice and plated in a six-well dish at 1 × 10⁶ cells/ml in 2 ml of lymphocyte media (RPMI 1640 supplemented with 10% FCS , 5 mM l-Alanyl-l-Glutamine , 100 IU/ml penicillin–streptomycin and 2 mM β-mercaptoethanol with 5 ng/ml of Daedalus IL-7 or commercial IL-7 .
Media with IL-7 was replaced at Day 3 and the resulting cell populations were evaluated by fluorescence-activated cell sorting (FACS) at Day 5.
Cells were stained with fluorescein isothiocyanate (FITC) anti-mouse IgM and PE anti-mouse B220 (BD) in FACS staining buffer (PBS with 2.5% FCS) and analyzed on an LSRII flow cytometer (BD) and using FlowJo software .
Recombinant human LIF was tested for its ability to maintain pluripotency of murine ES cells.
Briefly, murine ES cells (AB1 ES cells kindly provided by Steve Jones, University of Massachusetts) were plated on irradiated feeder cells with either…

Bone marrow was harvested from C57BL/6 mice and plated in a six-well dish at 1 × 10⁶ cells/ml in 2 ml of lymphocyte media (RPMI 1640 supplemented with 10% FCS , 5 mM l-Alanyl-l-Glutamine , 100 IU/ml penicillin–streptomycin and 2 mM β-mercaptoethanol with 5 ng/ml of Daedalus IL-7 or commercial IL-7 .
Media with IL-7 was replaced at Day 3 and the resulting cell populations were evaluated by fluorescence-activated cell sorting (FACS) at Day 5.
Cells were stained with fluorescein isothiocyanate (FITC) anti-mouse IgM and PE anti-mouse B220 (BD) in FACS staining buffer (PBS with 2.5% FCS) and analyzed on an LSRII flow cytometer (BD) and using FlowJo software .
Recombinant human LIF was tested for its ability to maintain pluripotency of murine ES cells.
Briefly, murine ES cells (AB1 ES cells kindly provided by Steve Jones, University of Massachusetts) were plated on irradiated feeder cells with either…

Erythroid differentiation was carried out on MS-5 stromal cells as previously described ⁴ cells were plated in 2 mL medium'>serum-free expansion medium containing 100 ng/ml SCF, 5 ng/ml IL-3, 3 U/ml erythropoietin (EPO), and 1 mM hydrocortisone and cultured in 6-wells plates at 37°C and 5% CO₂ for 4 days.
The cells were then diluted into 6 ml of the same medium in a 25 cm² flask and cultured for an additional 4 days.
Expanded cells were washed with basal medium and resuspended in 25 ml of basal medium with 3 U/ml EPO and plated onto a newly confluent 75 cm² flask of MS-5 feeder cells and cultured for 3 days.
For final maturation, erythroid cells were co-cultured on feeder cells for 10 days using basal medium without cytokines with one medium change after 5 days at which time 2×10⁶

Erythroid differentiation was carried out on MS-5 stromal cells as previously described ⁴ cells were plated in 2 mL medium'>serum-free expansion medium containing 100 ng/ml SCF, 5 ng/ml IL-3, 3 U/ml erythropoietin (EPO), and 1 mM hydrocortisone and cultured in 6-wells plates at 37°C and 5% CO₂ for 4 days.
The cells were then diluted into 6 ml of the same medium in a 25 cm² flask and cultured for an additional 4 days.
Expanded cells were washed with basal medium and resuspended in 25 ml of basal medium with 3 U/ml EPO and plated onto a newly confluent 75 cm² flask of MS-5 feeder cells and cultured for 3 days.
For final maturation, erythroid cells were co-cultured on feeder cells for 10 days using basal medium without cytokines with one medium change after 5 days at which time 2×10⁶ cells were harvested for nucleic acid purification.
T-cell differentiation was carried on OP9 stromal cells as previously described ⁴ nucleofected cells were resuspended in αMEM with 20% FBS , 5 ng/ml IL-7, and 5 ng/ml Flt3L (both, , ) onto sub-confluent OP9-DLL1 cells.
Fresh media with cytokines was replaced every 3 to 4 days.
B-cell differentiation was carried out on MS-5 stromal cells as reported ⁵ nucleofected CD34⁺ cells were resuspended in 15 ml of αMEM with 10% FBS, 10 ng/ml SCF, and 10 ng/ml G-CSF and seeded onto a confluent MS-5 layer in a 75 cm² flask.
Ten milliliters of fresh medium was added after 7 days and cells were maintained with twice weekly half-media changes.
Myeloid and granulocyte differentiations were performed on MS-5 stromal cells as described ⁵ nucleofected CD34⁺ cells were seeded in 1 ml IMDM with 10% FBS, 2 mM L-alanyl-glutamine, 50 ng/ml SCF, and 50 ng/ml IL-3 in six-well plates.
After 3 days in culture, cells were washed and seeded into 25 cm² flasks in basal medium plus 50 ng/ml SCF, 50 ng/ml IL-3, and 10 ng/ml G-CSF.
Cells were maintained at 2×10⁵ to 8×10⁵ cells per ml in complete medium with cytokines for 10 days until harvest.

For cytokine signalling, LN T cells (5 × 10⁶ cells/ml) were cultured 16 h with recombinant mouse IL-7 (10 ng/ml), IL-4 (40 ng/ml), IL-6 (45 ng/ml& ) or IL-15 (100 ng/ml& ) as previously described (

T cells co-express CD200 and CD200R following TCR activation.

C. Naïve LN cells were stimulated with anti-CD3 (1 µg/ml) + anti-CD28 (2 µg/ml) either transiently on Ab-coated wells for the first 2d then removed to fresh media (blue lines) or chronically with Ab-coated aAPC present throughout the culture (red lines) in Th1 (top row), Th2 (middle row) or non-polarising (IL-7, bottom row) conditions.

IL-7 mediated growth and survival is dose dependent in vitro.

CD8 T cells were enriched from C57Bl6/J donors and cultured with a range of IL-7 concentrations.

Reduced white adipose tissue mass and insulin sensitivity in IL-7 overexpressing mice.

Results are expressed as mean ± SEM of 7 WT mice (white bars) and 8 Tg IL-7 mice (black bars).

TGF-β is a negative regulator of NK cell generation CD11cdnR and wild-type mice were injected (i.p.)

Four days later, mice were sacrificed and HSC-enriched bone marrow cells were cultured in the presence of IL-7, SCF, and Flt3L.

In vitro Generation of NK cells

The generation of NK cells from stem cell precursors was conducted in vitro, based on protocol previously described.
Briefly, CD11c^dnR and wild-type mice were first treated (i.p.) with 5-FU (5-Fluorouracil) provided at 150 mg/kg body weight.
Four days later, mice were sacrificed and HSC-enriched bone marrow cells were cultured at 10,000 cells/well in complete RPMI supplemented with 0.5 ng/ml murine IL-7 , 30 ng/ml mouse SCF , and 100 U/ml murine Flt3L (eBiosciences).
Cells were re-fed with the same media on day 3.
On day 5, cultures were harvested and re-plated at 15,000/well on a confluent monolayer of OP9 stromal cells in complete RPMI containing 30 ng/ml murine IL-15 .
On day 8, cultures were re-fed with the same media with or without 5 ng/ml human TGF-β .
On day 11, cells were harvested and analyzed by flow cytometry.

Mouse Interleukin-7 Recombinant

Description

Methods

T cell activation assay

Functional assays

Functional assays

Differentiation of CD34+ cells

Functional assays

T cells co-express CD200 and CD200R following TCR activation.

IL-7 mediated growth and survival is dose dependent in vitro.

Reduced white adipose tissue mass and insulin sensitivity in IL-7 overexpressing mice.

TGF-β is a negative regulator of NK cell generation CD11cdnR and wild-type mice were injected (i.p.)

In vitro Generation of NK cells

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Mouse Interleukin-7 Recombinant

Description

Methods

T cell activation assay

Functional assays

Functional assays

Differentiation of CD34+ cells

Functional assays

T cells co-express CD200 and CD200R following TCR activation.

IL-7 mediated growth and survival is dose dependent in vitro.

Reduced white adipose tissue mass and insulin sensitivity in IL-7 overexpressing mice.

TGF-β is a negative regulator of NK cell generation CD11cdnR and wild-type mice were injected (i.p.)

In vitro Generation of NK cells

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