Total splenocytes were isolated from immunized mice as described above, and total lymphocytes were resuspended in complete IMDM medium (Gibco). To measure cytokine production, total splenocytes were treated with PMA (50 ng/mL) and ionomycin (1 μg/mL) for 4 hours at 37ºC and 5% CO2. GolgiPlug (BD Biosciences) was added to the culture medium (concentration = 1:1000) after 1 hour of culture. At the end of the stimulation period, cells were fixed and stained for flow cytometry using previously described methods (AUTHORS NEED TO BE QUERIED TO ADD THE CORRECT REFERENCE HERE). Notably, we used a specific clone of rat anti-mouse CXCR5 (clone L138D7, BioLegend) that can stain intracellular CXCR5 after paraformaldehyde fixation.
Complete imdm medium
Manufactured by Thermo Fisher Scientific
Sourced in Australia
Complete IMDM medium is a cell culture medium that provides a balanced combination of amino acids, vitamins, inorganic salts, and other essential components required for the growth and maintenance of various cell lines in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (1 mentions)
R595 lps, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
7 protocols using complete imdm medium
1
Cytokine Production Profiling of Activated Mouse Splenocytes
2
HuT78 CD4+ T Cell Culture and Lysis Protocol
3
Splenocyte Culture and Influenza Stimulation
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Single cell suspensions were prepared from spleen in complete IMDM medium (Gibco, Waltham, MA) with 10% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 5×10−5 M of 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Lymphocytes were counted and diluted to 5×106 cells/mL. 2.5×106 cells/well were added into 48-well plates. For lipopolysaccharides (LPS) stimulation wells, cells were cultured with 0.4 μg R595 LPS (Sigma-Aldrich, St. Louis, MO), and 100 μL of supernatant from concanavalin A-stimulated splenocytes, prepared as previously described [30 (link)]. Influenza virus-specific in vitro stimulation was performed as previously described [31 (link)]. Briefly, mouse splenocytes were cultured for 6 days with 10 μg β-propiolactone (BPL)-inactivated A/Cal09 (IRR, FR1184), H3N2 control antigen 2009–2013 (FR43), and B/Brisbane/08 (B/Bris08, FR 1188), respectively. The culture supernatants were harvested and stored at -80°C for mPlex-Flu assay. For the mPlex-Flu assay, all samples were thawed, diluted and assayed at same time to minimize batch effects.
4
HuT78 CD4+ T Cell Culture and Lysis Protocol
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HuT78 CD4+ T cells were acquired directly from ATCC (TIB-161). HuT78 CD4+ T cell lines were cultured at 37°C and 5% CO2 in complete IMDM medium (IMDM medium supplemented with 20% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, and 2mM l-glutamine) (Gibco). Retro/lentiviral transduced cell lines were kept in selection in the presence of 1-2 µg/mL puromycin. Jurkat E6.1 T cells were cultured in complete RPMI medium. For immunoblot analysis, the cells were lysed with the addition of 4-fold excess of hot 2X lysis buffer (20 mM Tris pH8.0, 2 mM EDTA, 2 mM Na3VO4, 20 mM DTT, 2% SDS, and 20% glycerol). Lysates were then heated to 95°C for 4 min and sonicated to reduce viscosity.
5
Flow Cytometry Analysis of Orthotopic Tumors
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For flow cytometry analysis orthotopic tumors were collected and minced into small pieces before digestion in Collagense IV (6000 U/ml) and DNase I (200 U/ml) for one hour at 37 ˚C. Cell suspension was filtered through a 40 µm cell strainer. For cytokine stainings, cells were restimulated with PMA (100 nM), ionomycin (1 µg/ml), and monensin (2 µg/ml) for 3–4 h at 37 ˚C in complete IMDM medium (Gibco). Viability staining was performed using the fixable viability dye eFluor780. Staining with fluorescent antibodies was carried out for 15 min at 4 ˚C in the dark. After washing in FACS buffer (2 mM EDTA, 2% FBS), cell suspension was acquired using BD Fortessa and FlowJo software (Treestar).
6
Antigen-Specific CD8+ T Cell Activation
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cDC1 and cDC2 were sorted from naïve lungs of WT or Akita mice. CD8+ T cells were obtained from splenocytes of T cell receptor-transgenic OT-I mice, in which all CD8+ T cells recognize the SIINFEKL peptide of OVA. Isolation was performed using CD90.2 MACS-bead (Miltenyi) pre-enrichment and subsequent flow cytometry-based sorting of CD8+CD11c− aufluorescent-negative cells. T cells and DC were then cocultured for 4 days in complete IMDM medium (Life Technologies) with 100 mg ml−1 OVA protein.
7
Antigen-Specific T Cell Activation
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BMDCs were obtained as described above. T cells were obtained with CD4 MACS-bead (Miltenyi) sorting from naïve splenocytes. Cells were then cultured together for 4 days in complete IMDM medium (Life Technologies) with the addition of varying concentrations of OVA323-339 peptide (Mimotopes Australia). Before flow cytometric analysis, cells were restimulated with PMA (Sigma-Aldrich) ionomycin (Sigma-Aldrich) and monensin (Sigma-Aldrich).
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