Human monocytes were isolated from buffy coats or fresh blood from healthy donors by serial density‐gradient centrifugation as previously described,23 or using the CD14 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, DE) accordingly to the manufacturer's instructions. Human blood cell isolation was approved by the institutional Ethical Committee of the Humanitas Clinical and Research Center (Authorization n° 2502, 09/04/2020 and Approval for the use of buffy coats issued on 28/01/2016). Monocytes were treated for 7 days with 25 ng/ml rhM‐CSF (Peprotech, London, GB) to obtain Mϕs, in RPMI 1640 (Lonza, Basel, CH) with 10% FBS (Lonza, Basel, CH) and 1% l ‐glutamine (Lonza, Basel, CH). Mϕs were activated in vitro by 18 h incubation with 100 ng/ml 055:B5 LPS (Sigma–Aldrich, St Louis, MO, USA), 20 ng/ml rhIFN‐γ (Peprotech, London, GB), a combination of 100 ng/ml LPS and 20 ng/ml rhIFN‐γ, 20 ng/ml rhTNF (Peprotech, London, GB), 20 ng/ml rhIL‐10 (Peprotech, London, GB), 20 ng/ml rh TGF‐β (rhTGF‐β) (Peprotech, London, GB), 10−6 M Dexamethasone (Dex) (MP‐Biomedicals, Illkirch, FR), 20 ng/ml rhIL‐4, or 10−6 M Dex in combination with 20 ng/ml IL‐4.
Rhifn γ
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
RhIFN-γ is a recombinant human interferon gamma product. It is a cytokine that plays a crucial role in immune response and inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Kynurenine, by Merck Group (4 mentions)
Rhtnf α, by Thermo Fisher Scientific (3 mentions)
Rhgm csf, by Thermo Fisher Scientific (2 mentions)
Rhil 1β, by Thermo Fisher Scientific (2 mentions)
Rhil 6, by Thermo Fisher Scientific (2 mentions)
Kynurenine, by Thermo Fisher Scientific (2 mentions)
Rapamycin, by Thermo Fisher Scientific (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Epacadostat, by MedChemExpress (2 mentions)
Rhm csf, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by MP Biomedicals (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Rhil 10, by Thermo Fisher Scientific (1 mentions)
Rhtnf, by Thermo Fisher Scientific (1 mentions)
Cd14 microbead kit, by Miltenyi Biotec (1 mentions)
L glutamine, by Lonza (1 mentions)
Ficoll paque, by STEMCELL (1 mentions)
20 protocols using rhifn γ
1
Generation and Activation of Human Macrophages
2
Generation of Large Multinucleated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood from healthy volunteers was collected after informed consent and ethical approval. Peripheral blood mononuclear cells (PBMCs) are isolated through Ficoll-Paque™ (Stemcell, USA) density gradient centrifugation. Wash the PBMCs with PBS. PBMCs were plated at a density of 106 cells/mL in RPMI-1640 medium containing 2% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin. After an hour, the cells were washed for three times with PBS and the adherent cells were cultured in the presence of 20 ng/mL rhGM-CSF (Peprotech, USA) and 10 ng/mL rh IFN-γ (Peprotech, USA) in RPMI-1640 medium containing 20% FBS for 7 days.11 (link) The cells were supplemented with fresh medium every 2 days. At the end of the culture period, the number of nuclei per cell was determined by rapid Wright-Giemsa staining (Jiancheng Biotech, China) or immunofluorescence staining. LGCs were defined as cells with more than three nuclei per cell.
3
Peripheral Blood Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh peripheral blood samples were collected from healthy volunteers. The human lung cancer cell line A549 was derived from the passaged cell line preserved in the immunology laboratory of Zunyi Medical University. Tim-3 blocking antibody (AB_1877089) and PD-1 blocking antibody (AB_2820104) were purchased from Biolegend Inc., USA. The cell factors rhIL-4, rhGM-CSF, rhIFN-γ, rhTNF-α, and CD3McAb were purchased from Peprotech Inc., USA. Flow cytometry antibodies CD3-PE-Cy5, CD56-RD1, CD4-FITC, CD8-PC-Cy7, Tim-3-APC, and PD-1-PE were purchased from BD Inc., USA. The 1640 medium was purchased from Thermo Scientific Inc., USA. The GTT-551 H3 medium was from TaKaRa Company, Dalian, China.
4
In Vitro B Cell Differentiation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro naive and memory B cell differentiation assays were performed as reported previously (8 (link), 13 (link)). In short, irradiated 3T3 fibroblasts expressing human CD40L were cocultured with purified naive mature (CD19+CD38–/dimCD27–; primary response) or memory (CD19+CD38–/dimCD27+; recall response) B cells in the presence of rhIL-21 (50 ng/ml; Thermo Fisher Scientific). For naive B cell cultures, rhIFN-γ (50 ng/ml; Peprotech) was added with and without CpG-ODN (10 μg/ml; Invivogen) to induce class switching and ASC development, respectively (8 (link)). After 6 (recall response) or 11 (primary response) days of culturing, viable (live/dead–) CD19+ cells were examined using flow cytometry. The supernatants were collected and stored at –80°C until further use.
5
Isolation and Characterization of CIK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CIK cells were obtained from PBMCs of healthy donors isolated by means of Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, according to standard protocols.10 (link) PBMCs were plated in RPMI 1640 (Euroclone) supplemented with 10% heat-inactivated FBS (Gibco), 1% Ultraglutamine, 1% Hepes buffer, 1% penicillin/streptomycin (all from Lonza), at 37°C and 5% CO2, and stimulated with rhIFN-γ (PeproTech) at 1000 U/ml at day 0. Twenty-four hours later, anti-CD3 mAb (OKT-3, Ortho Biotech Inc) at 50 ng/ml and rhIL-2 (Proleukin, Novartis) at 500 IU/ml were added to the culture medium; every 2–3 days medium was replenished and fresh rhIL-2 at 500 IU/ml was added. CIK cells phenotype was analyzed by multi-color flow cytometry, using the following antibodies: CD3-BV510 (clone UCHT1), IL-2-BV421 (clone 5344.111), CD25-APC (IL-2 Rα, clone M-A251), CD122-BV650 (IL-2Rβ, clone Mik-β3), CD132-BV786 (IL-2Rγ, clone AG184), from BD Bioscience; CD56-PE (clone HCD56), CD16a-FITC (clone 3G8), from BioLegend. Flow cytometry analysis was performed on either LSRII or Celesta, using DIVA software (BD Bioscience). Data analyzes were performed using FlowJo software (Treestar).
6
MHC-I and MHC-II Induction in Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Overall, 100,000 tumor cells were cultured in T2 medium containing the specified stimulation agents. For MHC-I upregulation, cells were treated with 10 ng/mL recombinant human Interferon Gamma (rhIFN)-γ (Peprotech) for 24 hours. For MHC-II induction, cells were incubated with 30 ng/mL rhIFN-γ for 3 days or transduced with human Class II Major Histocompatibility Complex Transactivator (hCIITA)+/- lentiviral vectors. The appropriate controls of unstimulated and untransduced cells were also included in the experiments. MHC-I and/or MHC-II expression was detected by flow cytometry using the antibodies specified on online supplemental table 2 .
hCIITA was cloned into pHIV-Luc-ZsGreen (Addgene) backbone and DNA was isolated using EndoFree Plasmid Kit (Qiagen). hCIITA-expressing lentiviral particles were produced by co-transfecting HEK293T cells with the pHIV-CIITA-ZsGreen plasmid, the pMD2.G envelope plasmid and the psPAX2 packaging plasmid. Virus-containing supernatants were concentrated by ultracentrifugation and TCLs were transduced by spinoculation with hCIITA-expressing lentiviral particles.
hCIITA was cloned into pHIV-Luc-ZsGreen (Addgene) backbone and DNA was isolated using EndoFree Plasmid Kit (Qiagen). hCIITA-expressing lentiviral particles were produced by co-transfecting HEK293T cells with the pHIV-CIITA-ZsGreen plasmid, the pMD2.G envelope plasmid and the psPAX2 packaging plasmid. Virus-containing supernatants were concentrated by ultracentrifugation and TCLs were transduced by spinoculation with hCIITA-expressing lentiviral particles.
7
Cell Growth Modulation by IFN-γ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell growth rates were determined using a Cell Counting Kit-8 (DOjinDO Laboratories, Japan) (CCK-8). Cells were seeded in a 96-well plate at a density of 5 × 103 cells and incubated at 37 °C in a humidified atmosphere containing 5 % CO2. After 24 h, cultured cells were treated with different dosages of recombinant human IFN-γ (rhIFN-γ) (Peprotech, Rocky Hill, NJ, USA). The CCK-8 assay was then performed according to the manufacturer’s instructions. Three independent experiments were performed, and data were expressed as the mean ± SD. For the focus formation assay, 1 × 103 cells were seeded in a 6-well plate and stimulated with or without 10 ng/ml(10000 IU/ml) rhIFN-γ for 2 weeks. Media were changed every three days. Surviving colonies were fixed and stained with 1 % crystal violet. Three independent experiments were performed.
8
IFN-γ Stimulated THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP-1 cells with a density of 4 × 105 cells/mL were cultured for 4 h in DMEM medium containing 0.3% FBS, and stimulated by 80 ng/mL of rhIFN-γ (Peprotech, Rocky hill, CT, USA) for 48 h43 (link). The cells were then harvested and incubated with human HLA-DR flow antibody (eBioscience, San Diego, CA, USA) for 30 min at 4 °C and the cells were washed and analyzed by a FACS Calibur flow cytometry (BD Bioscience, San Jose, CA, USA).
9
Temporal Response of Intestinal Epithelium to Microbial Challenges
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C2BBe1 cells were seeded in 24-well plates at a density of 1 × 105 cells/well in full DMEM culture medium. Once cells reached confluency, they were cultured in serum and antibiotic-free DMEM overnight (12–16 h) followed by co-culture for 3 h with the commensal E. coli K12, the CD-pathobiont AIEC, the pathogen S. typhimurium [all bacteria strains added at MOI 10:1] as recently described (27 (link)), or a pro-inflammatory cytokine cocktail [consisting of recombinant human (rh) TNF-α (20 ng/ml), rh IFN-γ (10 ng/ml), rh IL-1β (10 ng/ml), all purchased from Peprotech], followed by 3× wash in PBS + 1% Pen/Strep, and a further incubation in full culture DMEM media for 2 (T2), 5 (T5), and 13 (T13) h post infection/cytokine treatment. At each time point, cells were washed with PBS and lysates stored at −80°C for RT-qPCR analyses. All conditions were performed in triplicates.
10
Intracellular Cytokine Staining in Melanoma Cell Lines
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!