BM cells collected from 6- to 8-week-old C57BL/6 mice tibia and femur were flushed with PBS. Red blood cell (RBC) lysis buffer (BD Biosciences; CA, USA) was used to lyse red blood cells. Cells (2 × 106) were cultured at 37°C and 5% CO2 for 7 days in 6 cm dishes containing 3 ml Roswell Park Memorial Institute (RPMI)-1640 with 10% FBS, 1% streptomycin and penicillin, 1% MEM nonessential amino acids (NEAA) solution, 1% sodium pyruvate (Gibco, NY, USA), 2 μl 2-mercaptoethanol (Sigma-Aldrich, St. Louis, USA), 50 ng/ml GM-CSF (PeproTech), and 40 ng/ml interleukin (IL)-6 (PeproTech). The cells were harvested for testing or sorting by FACS Aria III (BD Biosciences, CA, USA) using CD11b-fluorescein isothiocyanate (FITC) (eBioscience, CA, USA) and Gr-1-PerCP Cy5.5 (eBioscience). Flow cytometry verified that all isolated MDSCs yielded >90% pure population. The purified MDSCs were then adoptively transferred to mice via the tail vein just before UUO. For cell tracing, some UUO mice were injected with MDSCs and stained with CFSE (Invitrogen).
Interleukin il 6
Manufactured by Thermo Fisher Scientific
Sourced in United States
Interleukin (IL)-6 is a cytokine produced by various cell types. It plays a key role in the immune response and inflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Cd11b fluorescein isothiocyanate fitc, by Thermo Fisher Scientific (1 mentions)
Gr 1 percp cy5, by Thermo Fisher Scientific (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Facsaria 3, by BD (1 mentions)
Rbc lysis buffer, by BD (1 mentions)
Ag490, by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Mouse lineage cell depletion kit, by Miltenyi Biotec (1 mentions)
Imdm medium, by Lonza (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
L glutamine, by Lonza (1 mentions)
5 protocols using interleukin il 6
1
Murine MDSC Isolation and Adoptive Transfer
2
Establishing Cell Culture Conditions
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The chemical library was obtained from the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI), where it is available to investigators for non-clinical research purposes. Information on compound synthesis and purity is not available from the DTP/NCI website. Interleukin (IL)-6 and epidermal growth factor (EGF) were obtained from PeproTech (Rocky Hill, USA). Cholera toxin, hydrocortisone, insulin, and the pan-JAK inhibitor AG-490 were purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals used were of analytical grade and were obtained from Sigma-Aldrich unless otherwise noted.
3
Isolation and Culture of Mouse Hematopoietic Stem Cells
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Lineage-negative cells were isolated from WT and Ackr2−/− BM using LS columns and the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec), according to the manufacturer’s instructions. Negative fraction was stained with Streptavidin-PB, Sca-1, cKit, CD34, and FcγRII/III antibodies and sorted. LSK were seed (1 × 103/well) in rounded-bottom 96-well plate in IMDM medium (Lonza) supplemented with 10% FCS (Sigma), 1% l -glutamine (Lonza), 20 ng/ml stem cell factor (SCF) (Peprotech), 10 ng/ml interleukin (IL)-6 (Peprotech), and 10 ng/ml IL-3 (Peprotech), as previously described54 (link). Cells were harvested 3 and 6 days after seeding, stained, and analyzed by flow cytometry.
4
CRISPR Gene Editing of Mobilized HSPCs
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Mobilized human CD34+ HSPCs (AllCells, Alameda, CA, USA) were thawed and cultured for 48 h at a density of 2.5 × 105 cells/mL in StemSpan SFEM II (StemCell Technologies, Vancouver, Canada), supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Biological Industries, Beit Haemek, Israel), stem cell factor (SCF), thrombopoietin (TPO), Fms-related tyrosine kinase 3 ligand (Flt3), and interleukin (IL)-6 (100 ng/mL each; PeproTech, Rocky Hill, NJ, USA). Cells were cultured at 37°C, 5% CO2, and 5% O2. 0.5 × 105 CD34+ HSPCs were reconstituted in P3 Primary Cell electroporation solution, according to the manufacturer’s instructions (Lonza, Basel, Switzerland) and mixed with RNP complexes at a final concentration of 1 or 4 μM. Cells were then supplemented with either 3.85 μM Alt-R EE (IDT, Coralville, IA, USA)14 (link) or an equivalent volume of PBS. The supplemented cell solution (final cell concentration of 2 × 106 /mL) was transferred into the Lonza 4D-Nucleofector and electroporated using the DZ-100 program. Recovered cells were cultured for 48–72 h prior to gDNA extraction with QuickExtract (Lucigen, Middleton, WI, USA). RNP complexes were formed and delivered in the same manner with the Alt-R HiFi Cas9 variant (IDT, Coralville, IA, USA).
5
Human Umbilical Cord Blood-Derived CD34+ Cell Culture
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Umbilical cord blood (UCB) was collected from the umbilical cord of full-term normal deliveries in collaboration with Milano Cord Blood Bank (IRCSS Ca’ Granda Foundation – Ospedale Maggiore Policlinico). The mononuclear cell fraction was obtained by density gradient centrifugation using Ficoll-Paque (Lymphoprep, Sentinel Diagnostics) and CD34+ HSPCs were immunomagnetically isolated using CD34 Microbead Kit (MiniMACS kit, Miltenyi Biotec). Flow-cytometric analysis allowed to assess the purity of sorted cell population, displaying 90% of CD34+ HSPCs and negligible presence of CD14+ (monocytes) and CD3+ (lymphocytes) cells (Additional file 1 : Figure S1). Isolated CD34+ HSPCs were cultured in Stem Span medium (StemCell Technologies) supplemented with 20 ng/mL of interleukin (IL)‐6 (PeproTech), 20 ng/mL of IL‐3 (PeproTech), 50 ng/mL of fms‐like tyrosine kinase 3 (FLT3, PeproTech), and 50 ng/mL of stem cell factor (SCF, PeproTech). Cells were cultured in HG (30 mM of glucose, Sigma-Aldrich) or NG (30 mM of mannitol, Sigma-Aldrich) conditions for up to 20 days and treated or not with increasing concentration of LIRA (50 nM and 100 nM; MedCHemExpress) ± selective GLP-1R antagonist exendin (9-39) (150 nM EXE; MedCHemExpress).
Capan-1 cells (HTB-79™), used as GLP-1R positive control, were purchased from ATCC and grown in RPMI medium supplemented with 20% FBS, as indicated by the supplier.
Capan-1 cells (HTB-79™), used as GLP-1R positive control, were purchased from ATCC and grown in RPMI medium supplemented with 20% FBS, as indicated by the supplier.
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