Mouse Interleukin-2 Recombinant

Mouse Interleukin-2 Recombinant

$70.00$2,500.00


accession P04351


Source Optimized DNA sequence encodingMouse Interleukin-2 mature chain was expressed in Escherichia Coli.
Molecular weight Native Mouse Interleukin-2, generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately17 kDa. Recombinant IL-2 is a monomeric protein consisting of150 amino acid residue subunits, and migrates as an approximately17 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >95%, as determined by SDS-PAGE and HPLC
Biological Activity The ED50 as determined by the dose dependent stimulation of murine CTLL-2 cells is <.1 ng/ml, corresponding to a specific activity of > x units/mg.
Protein Sequence MYSMQLASCV TLTLVLLVNS APTSSSTSSS TAEAQQQQQQ QQQQQQHLEQ LLMDLQELLS RMENYRNLKL PRMLTFKFYL PKQATELKDL QCLEDELGPL RHVLDLTQSK SFQLEDAENF ISNIRVTVVK LKGSDNTFEC QFDDESATVV DFLRRWIAFC QSIISTSPQ
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation Recombinant mouse IL-2 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.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 at° -° 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.


Biological Process Immunity
Molecular function Cytokine
Molecular function Growth-factor

Methods

CTL and NK cell assays

  • Splenic mononuclear cells were prepared from individual mice one week after immunization.
  • The cells were cultured at 37°C for 2 h and non-adherent cells were used as NK effector cells.
  • To prepare CTL effector cells, splenic mononuclear cells (5×106) were co-cultured with 5×105 of mitomycin C (50 µg/mL, 40 min) inactivated B16 cells in 5 mL RPMI 1640 containing 10% FBS and 20 units/mL of recombinant IL-2 .
  • Five days later, the cells were harvested, purified by Ficoll gradient centrifugation and used as CTL effector cells.
  • The NK and CTL-mediated cytotoxicities were determined by lactate dehydrogenase (LDH) release assay.
  • Briefly, Yac-1 cells at 2×104/well were cultured in quadruplicate with the prepared NK at the ratios of 12.5, 25, and 50 effector to target cells, respectively in 5% FBS phenol-free RPMI 1640 in 96-well plates at 37°C for 4 h.…
  • Splenic mononuclear cells were prepared from individual mice one week after immunization.
  • The cells were cultured at 37°C for 2 h and non-adherent cells were used as NK effector cells.
  • To prepare CTL effector cells, splenic mononuclear cells (5×106) were co-cultured with 5×105 of mitomycin C (50 µg/mL, 40 min) inactivated B16 cells in 5 mL RPMI 1640 containing 10% FBS and 20 units/mL of recombinant IL-2 .
  • Five days later, the cells were harvested, purified by Ficoll gradient centrifugation and used as CTL effector cells.
  • The NK and CTL-mediated cytotoxicities were determined by lactate dehydrogenase (LDH) release assay.
  • Briefly, Yac-1 cells at 2×104/well were cultured in quadruplicate with the prepared NK at the ratios of 12.5, 25, and 50 effector to target cells, respectively in 5% FBS phenol-free RPMI 1640 in 96-well plates at 37°C for 4 h. The target cells or effector NK cells cultured in medium alone (negative) or treated with 1% triton X-100 (positive) were used as controls, respectively.
  • After centrifugation, their supernatants were harvested and the contents of LDH were determined using the CytoTox96 Non-Radioactive Cytotoxixity Assay kit, according to the manufacturer's instructions .
  • The similar procedures were performed for CTL assays using B16 or irrelevant syngeneic control hepa 1–6 cells as targets, respectively.
  • The cytotoxicity was calculated by the formula:

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  • Foxp3 DEREG mice were injected with 0.75 μg of DT in PBS i.p.
  • on days −2 and −1, and on days 2, 5, and 8 after RSV infection to induce and maintain Foxp3+ T-cell depletion.
  • IL-2 Cx were obtained by mixing 1 μg rmIL-2 and 5 μg anti-IL-2 ( eBioscience, San Diego, CA), and incubating at 37°C for 30 min.
  • Age- and sex-matched BALB/c or C57BL/6 mice received daily i.p.
  • injections of IL-2 Cx or PBS for three consecutive days.
  • In some experiments, mice were additionally injected i.p.
  • with 50 μl of rabbit anti-mouse asialo GM1 on days −2, 0, and 3 , or with 200 μl of anti-CD25 antibody (PC61; 1 mg ml−1) on days −1 and 3.

Cytokine Assays

  • ELISAs were also performed to assess levels of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ in the supernatant of the MLCs on day 4.
  • The capture monoclonal antibody (mAb) (JES5-2A5), detection mAb (JES5-16E3), and recombinant standard for IL-10 were from .
  • The capture and detection mAbs for IL-2 (JES6-1A12 and JES6-5H4, respectively), IL-4 (BVD-1D11 and BVD-24G2), and IFN-γ (R4-6A2 and XMG1.2) were from .
  • Recombinant standards for IL-2, IL-4, and IFN-γ were from .

Passive transfer of experimental autoimmune encephalomyelitis

  • Nine days post induction mice were killed and cells were obtained from the spleen and lymph nodes.
  • Cells were suspended in Roswell Park medium'>Memorial medium'>Institute medium supplemented with 10% fetal calf serum, 1 mM L-glutamine, antibiotics, MOG35-55 (20 μg/mL) and mouse recombinant IL-2 (50 units/mL, , , ) and incubated for three days in a CO2 humidified incubator.
  • Cells were harvested and live cells were separated with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and injected into naïve mice.
  • In control groups of cells extracts, cells were frozen after separation, thawed three times and then injected into naïve mice.
  • On the day of inoculation and 48 hours later, 100 ng of pertussis toxin in 0.1 mL saline was also administered by intraperitoneal injection.
  • Mice were followed for clinical symptoms of EAE and evaluated according to a 0 to 6 point score (0 represents no clinical signs and 6…
  • Nine days post induction mice were killed and cells were obtained from the spleen and lymph nodes.
  • Cells were suspended in Roswell Park medium'>Memorial medium'>Institute medium supplemented with 10% fetal calf serum, 1 mM L-glutamine, antibiotics, MOG35-55 (20 μg/mL) and mouse recombinant IL-2 (50 units/mL, , , ) and incubated for three days in a CO2 humidified incubator.
  • Cells were harvested and live cells were separated with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and injected into naïve mice.
  • In control groups of cells extracts, cells were frozen after separation, thawed three times and then injected into naïve mice.
  • On the day of inoculation and 48 hours later, 100 ng of pertussis toxin in 0.1 mL saline was also administered by intraperitoneal injection.
  • Mice were followed for clinical symptoms of EAE and evaluated according to a 0 to 6 point score (0 represents no clinical signs and 6 represents death as a result of disease).

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2.7. Mixed Leukocyte Culture and Cytokine Assays

  • An ELISA was also performed to assess levels of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ in the supernatant of the MLC on day 4.
  • The capture mAb (JES5-2A5), detection mAb (JES5-16E3), and recombinant standard for IL-10 were from .
  • The capture and detection mAbs for IL-2 (JES6-1A12 and JES6-5H4, resp.
  • ), IL-4 (BVD-1D11 and BVD-24G2, resp.
  • ), and IFN-γ (R4-6A2 and XMG1.2, resp.)
  • were from .
  • Recombinant standards for IL-2, IL-4, and IFN-γ were from .

Viral replication in CD4+ PBL cultures

  • To determine the efficiency of HIV-1 replication in peripheral blood CD4+ lymphocytes, we isolated CD4+ cells from buffy coat peripheral blood mononuclear cells (normal blood donors, Red Cross, Ghent, Belgium) by negative selection using paramagnetic beads (MACS , , ).
  • After isolation, the cells were cultured in medium'>RPMI medium supplemented with 2 mM L-glutamin, 10% heat-inactivated fetal calf serum, phytohemagglutinin (1 μg/mL , , ), 20 ng/mL IL-2 , 100 U/mL penicillin, and 100 g/mL streptomycin.
  • Thereafter, 1 ng of p24 antigen was added to 2,5*105 PBLs, and the culture was spinoculated at 2,300 rpm for 90 minutes at 32°C.
  • After centrifugation, the supernatant was removed and the cells were further cultured in RPMI supplemented with 20 ng/mL IL-2.
  • HIV replication was monitored at 1, 3, 5, 7, 9 and 11 days post-infection by measuring the amount of p24 antigen present in the supernatants, using a HIV p24 enzyme-linked immunosorbent…
  • To determine the efficiency of HIV-1 replication in peripheral blood CD4+ lymphocytes, we isolated CD4+ cells from buffy coat peripheral blood mononuclear cells (normal blood donors, Red Cross, Ghent, Belgium) by negative selection using paramagnetic beads (MACS , , ).
  • After isolation, the cells were cultured in medium'>RPMI medium supplemented with 2 mM L-glutamin, 10% heat-inactivated fetal calf serum, phytohemagglutinin (1 μg/mL , , ), 20 ng/mL IL-2 , 100 U/mL penicillin, and 100 g/mL streptomycin.
  • Thereafter, 1 ng of p24 antigen was added to 2,5*105 PBLs, and the culture was spinoculated at 2,300 rpm for 90 minutes at 32°C.
  • After centrifugation, the supernatant was removed and the cells were further cultured in RPMI supplemented with 20 ng/mL IL-2.
  • HIV replication was monitored at 1, 3, 5, 7, 9 and 11 days post-infection by measuring the amount of p24 antigen present in the supernatants, using a HIV p24 enzyme-linked immunosorbent assay kit .
  • Alternatively, we also determined the expression of the HSA reporter gene at each time point using flow cytometry, as was described before [

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CD4+ T cell cultures

  • CD4+ T cells were purified using DynaBeads FlowComp mouse CD4 kit and were activated on αCD3/αCD28 coated plates under the following culture conditions: TH1 conditions: αIL4 10μg/ml (11B11) 0.1μg/ml IFNγ , 10u/ml IL12, 40u/ml; TH2 conditions: αIL12 10μg/ml (Tosh, ), αIFNγ 10μg/ml (XMG1.2, ), 10ng/ml IL4 , 40u/ml; TH17 conditiions: IL-6 25ng/ml , TGFβ 2ng/ml , IL1β 10ng/ml , αIL4 10 μg/ml (11B11, ), αIFNγ 10μg/ml (XMG1.2, ), and αIL12 10μg/ml (Tosh, ).
  • IL2 (40u/ml) was added for the TH17 cultures in

Proliferation of CD4+ T cells cultured with stimulated or non-stimulated BMDCs.

mouse recombinant IL-2
  • CD4+ T cells were isolated from mesenteric lymph nodes of C57BL/6 mice, labeled with CFSE and co-cultured for 7 days with BMDCs (non-stimulated, stimulated with LPS or Trichinella spiralis antigens) in the presence of recombinant mouse IL-2 and anti-CD3 antibody.

Treg conversion assay

  • Previously harvested BMDCs (nonstimulated and stimulated with LPS or T.
  • spiralis antigens) were seeded in 96-well round-bottomed cell culture plates at 2.5 × 103 cells/well.
  • CD4+ CD25 T cells were added at 5 × 104 cells/well to obtain a final CD4+ CD25 T cell/BMDCs ratio of 20:1.
  • OVA (323–339) was added in each well to a final concentration of 0.2 μg/mL.
  • Co-cultivated cells were divided into three groups treated with: 40 μg/mL anti-TGF-β (111) antibody as negative control , 2 ng/mL TGF-β as positive control or 40 μg/mL of control mouse IgG .
  • On days 1 and 3 of cultivation, rhIL-2 was added in each well to a concentration of 5 ng/mL.
  • On day 4, cells were harvested and surface stained for CD4 and CD25, as well as intra-cellular stained for Foxp3 (eBioscience; according to manufacturer’s protocol), as described above.
  • Flow cytometry…
  • Previously harvested BMDCs (nonstimulated and stimulated with LPS or T.
  • spiralis antigens) were seeded in 96-well round-bottomed cell culture plates at 2.5 × 103 cells/well.
  • CD4+ CD25 T cells were added at 5 × 104 cells/well to obtain a final CD4+ CD25 T cell/BMDCs ratio of 20:1.
  • OVA (323–339) was added in each well to a final concentration of 0.2 μg/mL.
  • Co-cultivated cells were divided into three groups treated with: 40 μg/mL anti-TGF-β (111) antibody as negative control , 2 ng/mL TGF-β as positive control or 40 μg/mL of control mouse IgG .
  • On days 1 and 3 of cultivation, rhIL-2 was added in each well to a concentration of 5 ng/mL.
  • On day 4, cells were harvested and surface stained for CD4 and CD25, as well as intra-cellular stained for Foxp3 (eBioscience; according to manufacturer’s protocol), as described above.
  • Flow cytometry was performed on a FACSCalibur .
  • At least 10 000 gated events were acquired per sample, and data analysis was performed with FlowJo software.

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Cell analysis and transfection

  • For dendritic cell (DC) production, murine bone marrow cells were harvested from femurs of C57BL/6 (B6) mice and plated out in a single-cell suspension in 100ml medium'>RPMI medium (medium'>RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 1mM sodium pyruvate, 2mM L-glutamine, 25mM HEPES buffer, and 50µM 2-ME) in 6 well tissue culture plates (6ml/well).
  • GM- CSF was added at 20ng/ml and the cells incubated at 37°C for 7 d. NK cells were isolated from spleens taken from B6 mice using biotinylated anti-CD49b (DX5) antibody ( 553856) together with the CELLection Biotin Binder Kit ( 115–33D).
  • NK cells were resuspended at 2 x106 cells/ml in medium'>DMEM medium (medium'>DMEM with 10% FCS, 100U/ml penicillin and streptomycin, 1mM sodium pyruvate, 2mM glutamine, 50µM 2-ME) containing 1000U rIL-2 /ml (212–12) in a 96-well…
  • For dendritic cell (DC) production, murine bone marrow cells were harvested from femurs of C57BL/6 (B6) mice and plated out in a single-cell suspension in 100ml medium'>RPMI medium (medium'>RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 1mM sodium pyruvate, 2mM L-glutamine, 25mM HEPES buffer, and 50µM 2-ME) in 6 well tissue culture plates (6ml/well).
  • GM- CSF was added at 20ng/ml and the cells incubated at 37°C for 7 d. NK cells were isolated from spleens taken from B6 mice using biotinylated anti-CD49b (DX5) antibody ( 553856) together with the CELLection Biotin Binder Kit ( 115–33D).
  • NK cells were resuspended at 2 x106 cells/ml in medium'>DMEM medium (medium'>DMEM with 10% FCS, 100U/ml penicillin and streptomycin, 1mM sodium pyruvate, 2mM glutamine, 50µM 2-ME) containing 1000U rIL-2 /ml (212–12) in a 96-well U- bottomed plate and incubated at 37°C for 4 d.

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