Human Interleukin-7 Recombinant (E.Coli)

HomeHematopoietins, IL-2 family, Recombinant Human Cytokines

Human Interleukin-7 Recombinant (E.Coli)

$70.00 – $4,700.00

SKU: RKP13232 Categories: Hematopoietins, IL-2 family, Recombinant Human Cytokines Tags: il-7, il7, Interleukin-7, P13232

Description

Methods (19)


accession	P13232

Source	Optimized DNA sequence encoding Human Interleukin-7 mature chain was expressed in Escherichia Coli.
Molecular weight	Native human Interleukin-7 is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately17 kDa. Recombinant IL-7 is a monomeric protein consisting of153 amino acid residue subunits, and migrates as an approximately17 kDa protein under non-reducing conditions and reducing conditions in SDS-PAGE.
Purity	>97%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) determinedby dose-dependent stimulation of the proliferation of murineE8 cells is ≤.5 ng/ml, corresponding to a specific activity of ≥ x units/mg.
Protein Sequence	MFHVSFRYIF GLPPLILVLL PVASSDCDIE GKDGKQYESV LMVSIDQLLD SMKEIGSNCL NNEFNFFKRH ICDANKEGMF LFRAARKLRQ FLKMNSTGDF DLHLLKVSEG TTILLNCTGQ VKGRKPAALG EAQPTKSLEE NKSLKEQKKL NDLCFLKRLL QEIKTCWNKI LMGTKEH
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 Interleukin-7 was lyophilized from a.2 μm filtered PBS solution pH7.5.
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.

Interactor	P31785 IL2RG_HUMAN
Interactor	P16871 IL7RA_HUMAN
Molecular function	Cytokine
Molecular function	Growth-factor

Methods


IL-7 induces partial reactivation of latent HIV-1 in cultured TCM. Mock or DHIV latently infected, cultured TCM were incubated in the presence of IL-2, IL-7 or a combination of IL-2 and IL-7 (IL-2+IL-7) or costimulated with antibodies to CD3 and CD28 (αCD3/αCD28) and assessed for intracellular p24Gag by flow cytometry (1st Reactivation).
Splenic Treg and Tcon cells display different susceptibility to mitochondrial apoptosis. Cells were cultured either in medium for 9, 18 and 48 h or in the presence of 100 U/ml IL-2, 20 ng/ml IL-7, 1 μM SAHA, 100 ng/ml FasL, 10−8 M Dexamethasone, 10 μg/ml Etoposide and 100 nM Staurosporine for 18 h. For calculation of increased and relative survival cell viability was normalized to medium values.
Cell culture Sorted ILC subsets were cultured overnight in the presence of IL-7 and SCF R&D Systems; both at 10 ng/ml) in the presence or absence of PMA (50 ng/ml) and Ionomycin (100 ng/ml) or for 3 days with IL-7, SCF, and either IL-23 or IL-1b. Cells were cultured in 200 μl DMEM supplemented with 10% FCS . IL-17a and IL-22 production was measured using the uoSet ELISA development kit (&systems). ELISAs were performed according to the manufacturers’ instructions.
Cell culture Sorted ILC subsets were cultured overnight in the presence of IL-7 and SCF R&D Systems; both at 10 ng/ml) in the presence or absence of PMA (50 ng/ml) and Ionomycin (100 ng/ml) or for 3 days with IL-7, SCF, and either IL-23 or IL-1b. Cells were cultured in 200 μl DMEM supplemented with 10% FCS . IL-17a and IL-22 production was measured using the uoSet ELISA development kit (&systems). ELISAs were performed according to the manufacturers’ instructions.
Priming assays Cytotoxic T-lymphocytes were generated by loading Mel888 or MV-treated Mel888 onto DC at a ratio of 3:1. After 24 hours non-adherent C were harvested then cocultured with autologous PBMC at ratios varying from 1:15 to 1:45 in CTL media (PMI supplemented with 7.5% (v/v) human AB serum ( , ’s , ), 1% (v/v) L-glutamine, 1% v/v sodium pyruvate , 1% (v/v) non-essential amino acids, 1% (v/v) HEPES , 20μM β-mercaptoethanol and 5ng/ml IL-7 . IL-2 was added at 30IU/ml on day 4 only. CTL were restimulated after one week in an identical fashion.
Generation of CD8 T-cell clones. CD8 T-cell clones specific for PPI_3–11 were generated as described (_3–11 peptide in X-Vivo/5% AB serum for 1 h at 37°C. After extensive washing, matured DCs were cultured at a 1:30 ratio with freshly obtained immunomagnetically isolated CD8 T cells (CD8 Isolation Kit II ) in 48-well culture plates in X-vivo/5% AB serum supplemented with 10 ng/mL IL-7 , 0.1 ng/mL IL-15, 5% Cellkine , and 25 IU/mL IL-2 . On day 10, cells were harvested and stained with anti-CD3 (V500), anti-CD8 (APC H7), violet amine reactive dye (live/dead exclusion; pacific blue), and PPI_3–11 tetramer (PE and APC) as appropriate. CD3⁺CD8 double tetramer-positive T cells were flow sorted (for gating strategy, see Supplementary. 1) and seeded at one cell per well into 96-well plates containing 0.2 × 10⁶ irradiated PBMCs in culture medium (CM; X-Vivo/5% AB serum, with IL-7 [10 ng/mL], IL-15 [0.1 ng/mL], IL-2 [100 IU/mL], and 2.5%… CD8 T-cell clones specific for PPI_3–11 were generated as described (_3–11 peptide in X-Vivo/5% AB serum for 1 h at 37°C. After extensive washing, matured DCs were cultured at a 1:30 ratio with freshly obtained immunomagnetically isolated CD8 T cells (CD8 Isolation Kit II ) in 48-well culture plates in X-vivo/5% AB serum supplemented with 10 ng/mL IL-7 , 0.1 ng/mL IL-15, 5% Cellkine , and 25 IU/mL IL-2 . On day 10, cells were harvested and stained with anti-CD3 (V500), anti-CD8 (APC H7), violet amine reactive dye (live/dead exclusion; pacific blue), and PPI_3–11 tetramer (PE and APC) as appropriate. CD3⁺CD8 double tetramer-positive T cells were flow sorted (for gating strategy, see Supplementary. 1) and seeded at one cell per well into 96-well plates containing 0.2 × 10⁶ irradiated PBMCs in culture medium (CM; X-Vivo/5% AB serum, with IL-7 [10 ng/mL], IL-15 [0.1 ng/mL], IL-2 [100 IU/mL], and 2.5% Cellkine) and phytohemagglutinin-M (5 μg/mL). Growing CD8 T-cell clones were restimulated every 15–20 days. CMV-AYA clones were generated directly ex vivo after staining of PBMCs as above using CMV-AYA tetramers. Read more
Mixed lymphocytes tumor cell culture (MLTC) PBMCs isolated from melanoma patients were thawed and incubated overnight at 37°C. PBMCs (10⁶ cells/well) were cultured in the presence of autologous irradiated (150 Gy) melanoma cells at a lymphocyte to tumor ratio of 5:1 in 24-well plates with X-VIVO15 and 5% HS. Different cytokine combinations were added to these MLTCs in order to select the most suitable in vitro culture conditions in terms of T lymphocyte expansion and efficiency in tumor cell recognition: (1) 120 IU/ml rhIL-2 ; (2) 120 IU/ml rhIL-2 and 10 ng/ml rhIL-15 ; (3) 10 ng/ml rhIL-15; (4) 5 ng/ml rh-IL-7 ; (5) 120 IU/ml rhIL-2 and 5 ng/ml rhIL-7; (6) 120 IU/ml rhIL-2 and 10 ng/ml rhIL-21 ; (7) 10 ng/ml rhIL-21 alone. Fresh medium containing the above indicated cytokines was replaced every 3 days. These MLTCs were stimulated weekly with irradiated autologous melanoma cells, and their reactivity was tested starting from the 3rd week of culture.
CTL Induction Naïve CD8⁺ T cells were negatively isolated from PBMCs of A24-positive donors using the CD8⁺ T cell isolation kit II and anti-CD45RO microbeads . The isolated cells were more than 95% pure CD45RO⁻ CD8⁺ populations. These CD8⁺ T cells (1×10⁶ cells/well) were cocultured with irradiated (100 Gy) aAPCs (1×10⁵ cells/well) in 2 mL X-VIVO20 supplemented with 5% human AB serum (MP, , ) in wells of a 12-well plate in the presence of 10 ng/mL IL-12 . On day 3, 10 ng/mL IL-7 and IL-15 were added. Every 3 days, half the medium was exchanged for fresh medium containing 10 ng/mL IL-15. On day 12, the T cells were restimulated with γ-irradiated aAPCs. One day thereafter, IL-2 was added, to a final concentration of 20 U/mL. celltype'>To establish celltype'>T cell clones, a limiting dilution of the polyclonal Ccelltype'>TL was performed as… Naïve CD8⁺ T cells were negatively isolated from PBMCs of A24-positive donors using the CD8⁺ T cell isolation kit II and anti-CD45RO microbeads . The isolated cells were more than 95% pure CD45RO⁻ CD8⁺ populations. These CD8⁺ T cells (1×10⁶ cells/well) were cocultured with irradiated (100 Gy) aAPCs (1×10⁵ cells/well) in 2 mL X-VIVO20 supplemented with 5% human AB serum (MP, , ) in wells of a 12-well plate in the presence of 10 ng/mL IL-12 . On day 3, 10 ng/mL IL-7 and IL-15 were added. Every 3 days, half the medium was exchanged for fresh medium containing 10 ng/mL IL-15. On day 12, the T cells were restimulated with γ-irradiated aAPCs. One day thereafter, IL-2 was added, to a final concentration of 20 U/mL. celltype'>To establish celltype'>T cell clones, a limiting dilution of the polyclonal Ccelltype'>TL was performed as previously described Read more
Enhanced proliferation of T cell precursors on LmDL1-FL7., Lentivector constructs expressing mouse DL1, human IL-7 and human Flt3L. , qRT-PCR analysis for DL-1, Flt3L, and IL-7.
Hematopoietic Stem/progenitor Cell Cultures Stromal cells were irradiated (2000 rad) and plated (25,000 cells/cm²) on tissue culture treated wells in stroma media (see above) 24 hours before co-culture. HSPC were co-cultured with stroma (1000–5000 CD34+ cells/cm²) in “HSC media” (stroma media supplemented with human SCF (25 ng/ml), FLT-3L (25 ng/ml) and TPO (25 ng/ml) for 7–14 days before replating (100,000–200,000 cells/cm²). Half the media was replaced every other day. For B-cell differentiation, the expanded cells were co-cultured on irradiated OP9M2 in stroma media supplemented with human SCF (50 ng/ml), FLT-3L (40 ng/ml) and IL-7 (100 ng/ml) . Following expansion, cells were assayed by FACS and colony-forming assays (see below).