Mouse GM-CSF Recombinant

$70.00 – $2,900.00

SKU: RKP01587 Tags: CSF, csf2, Csfgm, GM-CSF, P01587

Description

Accession

P01587

Source

Optimized DNA sequence encodingMouse Granulocyte Macrophage-Colony Stimulating Factor mature chain was expressed in Escherichia Coli.

Molecular weight

Native Mouse Granulocyte Macrophage-Colony Stimulating Factor isgenerated by the proteolytic removal of the signal peptide and propeptide, this molecule has a calculatedmass of kDa. Recombinant mouse GM-CSF is a monomer protein, consisting of amino acidsandmigrates as an approximately14 kDa protein under reducing conditions.

Purity

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

Biological Activity

The ED50 as determined by the dose-dependent stimulation of the proliferation of murine FDC-P1 cells is ≤.1ng/ml, corresponding to a specific activity of ≥3 x units/mg.

Protein Sequence

MWLQNLLFLG IVVYSLSAPT RSPITVTRPW KHVEAIKEAL NLLDDMPVTL NEEVEVVSNE FSFKKLTCVQ TRLKIFEQGL RGNFTKLKGA LNMTASYYQT YCPPTPETDC ETQVTTYADF IDSLKTFLTD IPFECKKPGQ K

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 mouse GM-CSF was lyophilized from.2 μm filtered PBS solution, pH7.0.

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

In-vitro NK-DC co-culture

Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
The development of an NK-DC co-culture system required two independent steps.
Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14⁺ monocytes from PBMC.
Following isolation, CD14⁺ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO₂ cell culture incubator at 37°C for 5 days.
This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14⁻CD1a⁺ iDC cells, comparable with the published literature ⁺CD16⁺ NK cell.
Purities of >98% were attained consistent with published literature ₂ cell culture incubator at 37°C.
Co-culture cell ratios…

Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
The development of an NK-DC co-culture system required two independent steps.
Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14⁺ monocytes from PBMC.
Following isolation, CD14⁺ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO₂ cell culture incubator at 37°C for 5 days.
This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14⁻CD1a⁺ iDC cells, comparable with the published literature ⁺CD16⁺ NK cell.
Purities of >98% were attained consistent with published literature ₂ cell culture incubator at 37°C.
Co-culture cell ratios of 1∶1 and 1∶5 (NK∶DC) were then studied as these ratios are most favourable for DC maturation ⁶ of each cell type in a final media volume of 500 µl.
The control wells had either DC or NK cells in isolation.
Trans-well experiments were performed using the same conditions, but in the presence or absence of a 0.4 µm insert to separate NK cells and DC.
At the end of co-culture C were tested for the expression of the maturation markers C86 (, 2331 ) and HLA- and the chemokine receptor CC7 .
The DC gate on flow cytometry was defined by a combination of scatter plot and CD56 staining to identify NK cells.
The Δ Mean Fluorescence Intensity (Δ MFI) per experiment for CD86, HLA-DR and CCR7 was calculated as the difference in MFI for DC in co-culture with NK cells versus DC in isolation (background maturation).
Corresponding isotype controls were used.
Cells were analysed using a FACSCalibur flow cytometer with Winmdi 2.9 software , acquiring information from a total of 5,000 gated cells.

Tumor-specific IFN-γ release and cell-mediated cytotoxicity after vaccination with mGC8 cells and GM-CSF.

Vaccination with mGC8 with or without LP, GM-CSF, and IFA.

Tumor cell vaccination (prophylactic/therapeutic), LRAST

To determine the immunogenicity of the tumor cells, 10⁷tumor cells were irradiated with 10,000 rad and subcutaneously injected into mice.
Two weeks later, the mice were challenged by subcutaneous injection of 3 × 10⁶viable tumor cells into the opposite flank.
Experimental groups generally consisted of 5 mice.
Tumor development was followed by serial measurements of the tumor diameter and is depicted as tumor size (mm²) = d × D, where d and D were the shortest and the longest tumor diameter, respectively.
Animals were euthanized when D reached 10 mm.
Lymphopenia was induced by i.p.
injection of cyclophosphamide (200 mg/kg, , ).
This dose was chosen since earlier studies have shown an increased proliferation and long-term survival of antigen-specific T cells at this dose of cyclophosphamide, alone or in combination with fludarabine [⁷naïve syngeneic splenocytes followed by s.c. vaccination with irradiated mGC8 cells (10⁷, 10,000 rad) with or without a…

Bone marrow was extracted from the femur and tibia passed through a screen and washed in cold media.
2×10⁶ bone marrow cells were resuspended in 10 ml of DC media (RPMI, 10% LPS free FCS, β-mercaptoethanol, L-glutamine, 40 ng/ml GM-CSF and cultured in 10 cm petri dishes at 37°C.
On day 3, 10 ml of DC media was added to the culture.
On day 6 and 8, 10 ml of culture was removed, resuspended in 10 ml of fresh DC media and added back to the culture.
On day 9, BMDCs were resuspended in DC media⁻ (ex GM-CSF) at 2×10⁶cell/ml, re-plated in 24 well plates and stimulated with LPS (100 ng/ml), Poly I∶C (100 µg/ml), and/or IFNα (1000 u/ml).
On day 10, BMDCs were pulsed for 2 hours with gp33 peptide alone or gp33, gp276 and gp61 peptides (10⁻⁶ M) respectively, and washed prior to use in in…

Mouse bone marrow dendritic cells (BMDCs, CD11c⁺) were isolated from the bone marrow of BALB/c mice, according to the protocol described previously [₂.
Approximately 1 × 10⁶cells was added to each well of a six-well plate, and mouse GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) was added to the culture medium every other day.
After 6 days of culture, BMDCs were stimulated with C.
burnetii I Ag (10 μg/ml), Com1 (10 μg/ml), HspB (10 μg/ml), E.
coli LPS (2 μg/ml), or 25 μl elution buffer (mock pulse) for 24 h at 37°C and 5% CO₂.

Generation of BMDC and BMMC

Complete medium'>medium'>RPMI (cmedium'>medium'>RPMI), medium'>medium'>RPMI 1640 medium containing 10% fetal calf serum , was used as culture medium.
For BMDC induction, 5×10⁶ BM cells were cultured supplemented with 10 ng/ml recombinant murine GM-CSF for five days

GC frass-induced PAR-2 mRNA and cytokine production from BMDC.

Bone marrow was isolated from BALB/c mice and cultured in the presence of GM-CSF for 6 days.

GM-CSF induces CD103 expression on Flt3L-derived CD8α-equivalent DCs.

FACS analysis of developing DCs in bone marrow cells from C57BL/6, Batf3

Phagocytic cells assays

The monocyte/macrophage J774.3 cell line used in this study was kindly provided by Dr. María Inés Becker .
J774.3 cells were routinely grown in high-glucose medium'>DMEM medium (GIBCO), supplemented with 10% Fetal Bovine Serum and 1 mM HEPES (GIBCO) in T75 bottles.
Cells were incubated at 37°C and 5% CO₂ until 95% of confluence.
Before infection assays, cells were treated with 0.1 mg/ml trypsine for 5 min, recovered in 50 ml polypropylene tubes, and centrifuged at 1,800× g for 5 min at room temperature.
After three washes with supplemented DMEM medium, cell number and viability was determined in a haemocytometer, using the trypan blue staining (1 mg/ml).
5×10⁵ cells/ml were seeded in 24 well-plates and incubated overnight at 37°C and 5% CO₂.
DCs were prepared from bone marrow precursors of C57BL/6 mice.
Cells were incubated in complete RPMI 1640 medium supplemented with 5% FCS…

The monocyte/macrophage J774.3 cell line used in this study was kindly provided by Dr. María Inés Becker .
J774.3 cells were routinely grown in high-glucose medium'>DMEM medium (GIBCO), supplemented with 10% Fetal Bovine Serum and 1 mM HEPES (GIBCO) in T75 bottles.
Cells were incubated at 37°C and 5% CO₂ until 95% of confluence.
Before infection assays, cells were treated with 0.1 mg/ml trypsine for 5 min, recovered in 50 ml polypropylene tubes, and centrifuged at 1,800× g for 5 min at room temperature.
After three washes with supplemented DMEM medium, cell number and viability was determined in a haemocytometer, using the trypan blue staining (1 mg/ml).
5×10⁵ cells/ml were seeded in 24 well-plates and incubated overnight at 37°C and 5% CO₂.
DCs were prepared from bone marrow precursors of C57BL/6 mice.
Cells were incubated in complete RPMI 1640 medium supplemented with 5% FCS , 2 mM glutamine, 1 mM non-essential amino acids, 1 mM pyruvate, 1 mM HEPES, and 10 ng/ml of recombinant murine GM-CSF .
All cell culture media were acquired from GIBCO .
Culture media was replaced every 2 days.
After 6 days, the phenotype of DCs was analyzed by flow cytometry for the expression of the surface markers CD11c, CD86 and CD40, which revealed over 70% CD11c⁺ with an immature phenotype.
Before the infection assays, DCs were washed three times with PBS and then culture media was replaced with complete medium'>RPMI medium without antibiotics.
DCs and macrophages were infected with S. Enteritidis PT1 at a multiplicity of infection (MOI) equal to 25.
The MOI was confirmed by plating serial dilutions of bacterial cultures on LB agar.
After 1 h of incubation at 37°C and 5% CO₂, the supernatant of infected cells was recovered and stored for genomic DNA preparation.
Cells were washed two times with PBS and 1 ml of appropriate medium supplemented with gentamicin 50 mg/ml was added to cell cultures to kill the remaining extracellular bacteria.
After 2, 18 and 24 h of infection, cells were removed from the wells and centrifuged at 783× g for 5 min.
No significant changes on cell viability were observed after infection with S. Enteritidis at the time points used on the experiments (data not shown).
To recover intracellular bacteria, phagocytic cells were treated with 1 ml of lysis solution (19% ethanol, 0.1% SDS, 1% saturated basic phenol) for 30 min on ice.
After the incubation period, the cell lysate was centrifuged at 7,043× g for 5 min, and genomic DNA was extracted following the methodology mentioned above.
In order to quantify the amount of intracellular bacteria at different time points, either 100,000 DCs or 10,000 J774.3 cells were treated with 1 ml of PBS-0.1% triton X-100 for 15 min at room temperature.
100 µl of cell lysates were plated on LB agar and the plates were incubated at 37°C for 18 h. In parallel, an equal amount of bacteria used to infect cells was incubated in 1 ml of cell medium.
The incubation was performed for 2 h and after that time bacteria were recovered by centrifugation at 7.043× g for 5 min, and genomic DNA was prepared as described above.

Mouse BMDCs were generated from bone marrow progenitor cells, according to the modified protocol of[

Mouse GM-CSF Recombinant

Description

Methods

In-vitro NK-DC co-culture

Tumor-specific IFN-γ release and cell-mediated cytotoxicity after vaccination with mGC8 cells and GM-CSF.

Tumor cell vaccination (prophylactic/therapeutic), LRAST

BMDC culture

Immunoprotection analysis of Com1- and HspB-pulsed BMDCs

Generation of BMDC and BMMC

GC frass-induced PAR-2 mRNA and cytokine production from BMDC.

GM-CSF induces CD103 expression on Flt3L-derived CD8α-equivalent DCs.

Phagocytic cells assays

Generation of bone marrow-derived DCs

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Mouse GM-CSF Recombinant

Description

Methods

In-vitro NK-DC co-culture

Tumor-specific IFN-γ release and cell-mediated cytotoxicity after vaccination with mGC8 cells and GM-CSF.

Tumor cell vaccination (prophylactic/therapeutic), LRAST

BMDC culture

Immunoprotection analysis of Com1- and HspB-pulsed BMDCs

Generation of BMDC and BMMC

GC frass-induced PAR-2 mRNA and cytokine production from BMDC.

GM-CSF induces CD103 expression on Flt3L-derived CD8α-equivalent DCs.

Phagocytic cells assays

Generation of bone marrow-derived DCs

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