1 × 105 tumor cells per well were cultured in 96-well plates (flat bottom plate for adherent cells, round bottom plate for suspension cells) at 37 °C 5% CO2. 7C6 (a human IgG1 mAb kindly provided by Dr Kai W. Wucherpfennig) or human IgG1 isotype control (Biolegend, Koblenz, Germany) antibody were added at 10 µg/mL. After 24 h of culture, MICA and MICB on cell surface were detected following staining with APC conjugated anti-MICA/B antibody or IgG2a isotype control. For detaching adherent cells without disturbing the integrity of surface molecule, Accutase (Biolegend, Koblenz, Germany) was used. Prior to the staining process, Fc receptors were blocked with Human TrueStain FcX™ (Biolegend, Koblenz, Germany) at a final dilution of 1:100. Hoechst 33258 (Cayman Chemical, Hamburg, Germany) was added before flow cytometry analysis for viable cells gating. Samples were acquired using FACS Canto II (BD).
Igg2a isotype control
Manufactured by BioLegend
Sourced in United States
The IgG2a isotype control is a laboratory tool used to establish baseline binding levels and to evaluate non-specific binding in flow cytometry and other immunoassay applications. It serves as a negative control by providing a reference point for distinguishing antigen-specific signal from background noise.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Human truestain fcx, by BioLegend (1 mentions)
Hoechst 33258, by Cayman Chemical (1 mentions)
Accutase, by BioLegend (1 mentions)
Human igg1 isotype control, by BioLegend (1 mentions)
α gapdh, by Cell Signaling Technology (1 mentions)
α flag m2, by Merck Group (1 mentions)
α histone h3, by Cell Signaling Technology (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Il 17a, by R&D Systems (1 mentions)
Il 17f, by R&D Systems (1 mentions)
Round bottom 96 well plate, by Greiner (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Etomoxir, by Merck Group (1 mentions)
8 protocols using igg2a isotype control
1
Detecting MICA/B on Tumor Cells
2
Blocking CD56 Expression on NK Cells During A. fumigatus Infection
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A GPR165 (IgG2a) monoclonal blocking antibody was kindly provided by Daniela Pende and Alessandro Moretta and CD56 was blocked as previously described53 . NK cells (4 × 106 cells/ml) were incubated in CD56 blocking antibody (10.9 μg/ml) or IgG2a isotype control (Biolegend, 10.9 μg/ml) diluted in RPMI + FCS for 30 min at 37 °C. Afterwards, NK cells were cultured alone or in co-culture with A. fumigatus germ tubes (MOI 0.5) at a NK cell concentration of 1 × 106 cells/ml. CD56 blocking antibody and isotype control were 4 fold diluted during culture. After 9 h, cells were harvested and CD56 and CD69 expression was evaluated by flow cytometry. Supernatants of co-cultures were stored at −20 °C until a ProcartaPlexTM or ELISA was performed.
3
Monoclonal Antibody Production and Characterization
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The mouse mAb S16 was produced by Sugawara et al.27 (link). BALB/c mice were immunized with 50 μg purified HEF proteins of C/Ann Arbor/1/50 in Freund’s complete adjuvant thrice at weekly intervals. The hybridoma cells secreting antibodies to HEF were screened using enzyme-linked immunosorbent assay (ELISA). The isotype of S16 was classified as IgG2a using double immunodiffusion.
For western blotting, α-mouse ACAA2 (ab128911; Abcam, Cambridge, UK), α-GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA), α-HSP60 (#12165; Cell Signaling Technology), α-histone H3 (#4499; Cell Signaling Technology), α-Flag M2 (F1804; Sigma-Aldrich, St. Louis, MO, USA), and α-mouse albumin (#4929; Cell Signaling Technology) polyclonal antibodies (pAbs) were used as the primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse and rabbit IgG were purchased from Bio-Rad (Hercules, CA, USA). The pAb against mouse ACAA2 (TA321746; OriGene, Rockville, MD, USA) and HRP-conjugated goat α-mouse IgG2a antibody (Southern Biotech, Birmingham, AL, USA) were used for ELISA. The IgG2a isotype control (BioLegend, San Diego, CA, USA) was used as a control antibody for the indirect immunofluorescence assay and injection of S16 into mice.
For western blotting, α-mouse ACAA2 (ab128911; Abcam, Cambridge, UK), α-GAPDH (#5174; Cell Signaling Technology, Danvers, MA, USA), α-HSP60 (#12165; Cell Signaling Technology), α-histone H3 (#4499; Cell Signaling Technology), α-Flag M2 (F1804; Sigma-Aldrich, St. Louis, MO, USA), and α-mouse albumin (#4929; Cell Signaling Technology) polyclonal antibodies (pAbs) were used as the primary antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse and rabbit IgG were purchased from Bio-Rad (Hercules, CA, USA). The pAb against mouse ACAA2 (TA321746; OriGene, Rockville, MD, USA) and HRP-conjugated goat α-mouse IgG2a antibody (Southern Biotech, Birmingham, AL, USA) were used for ELISA. The IgG2a isotype control (BioLegend, San Diego, CA, USA) was used as a control antibody for the indirect immunofluorescence assay and injection of S16 into mice.
4
Evaluating B4 mAb Therapy in Murine MM
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C57BL/KaLwRij mice were injected to the tail vein with 1 × 106 5TGM1 murine MM cells. After 14 days, the mice were separated into 2 or 3 groups and administered either B4 mAb or the IgG2a isotype control (BioLegend) at doses between 30 μg and 200 μg every other day into the tail vein, for a total of 5 injections or 11 injections for long-term studies.
5
Inflammatory Responses to Bacterial LPS Stimuli
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P. gingivalis LPS, Pam2CSK4, Escherichia coli (0111: B4 strain) LPS (InvivoGen, San Diego, CA, USA), IL-4, IL-17A, IL-17F, and IFN-γ (R&D SYSTEMS, Minneapolis, MN, USA) were used for stimulation. Pam2CSK4 and E. coli LPS were added to cell cultures at a final concentration of 1.0 µg/ml. IL-4, IL-17A, IL-17F, and IFN-γ were added at a final concentration of 10 ng/ml. IgG2a isotype control (BioLegend, San Diego, CA, USA), TLR2-neutralizing antibody (anti-TLR2), and TLR4-neutralizing antibody (anti-TLR4) (IMGENEX, San Diego, CA, USA) were used to block the biological activity of TLRs; they were added at a final concentration of 10 ng/ml 30 min prior to stimulation with P. gingivalis LPS, Pam2CSK4, and E. coli LPS. For P. gingivalis LPS stimulation, various doses of LPS (0.0001, 0.001, 0.01, 0.1, and 1.0 µg/ml) were added to the THP-1 cell and PMA-treated cell culture and stimulated for various time periods (1, 2, 4, 12, 24, 48, and 72 h). MG-132 (Calbiochem, Darmstadt, Germany), a specific inhibitor of NF-κB, was added at a final concentration of 5.0 ng/ml 1 h prior to stimulation with P. gingivalis LPS.
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P. gingivalis LPS, Pam2CSK4, Escherichia coli (0111: B4 strain) LPS (InvivoGen, San Diego, CA, USA), IL-4, IL-17A, IL-17F, and IFN-γ (R&D SYSTEMS, Minneapolis, MN, USA) were used for stimulation. Pam2CSK4 and E. coli LPS were added to cell cultures at a final concentration of 1.0 µg/ml. IL-4, IL-17A, IL-17F, and IFN-γ were added at a final concentration of 10 ng/ml. IgG2a isotype control (BioLegend, San Diego, CA, USA), TLR2-neutralizing antibody (anti-TLR2), and TLR4-neutralizing antibody (anti-TLR4) (IMGENEX, San Diego, CA, USA) were used to block the biological activity of TLRs; they were added at a final concentration of 10 ng/ml 30 min prior to stimulation with P. gingivalis LPS, Pam2CSK4, and E. coli LPS. For P. gingivalis LPS stimulation, various doses of LPS (0.0001, 0.001, 0.01, 0.1, and 1.0 µg/ml) were added to the THP-1 cell and PMA-treated cell culture and stimulated for various time periods (1, 2, 4, 12, 24, 48, and 72 h). MG-132 (Calbiochem, Darmstadt, Germany), a specific inhibitor of NF-κB, was added at a final concentration of 5.0 ng/ml 1 h prior to stimulation with P. gingivalis LPS.
6
PBMCs Stimulation and Cytokine Analysis
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Isolation of PBMCs was performed by differential centrifugation over Ficoll-Paque (GE Healthcare). Cells were adjusted to 5 × 106 cells/mL (Beckman Coulter) and suspended in RPMI 1640 (Gibco) supplemented with 10 μg/mL gentamicin (Lonza), 10 mM L-glutamine (Life Technologies), and 10 mM pyruvate (Life Technologies). 100 μL of PBMCs was incubated in round-bottom 96-well plates (Greiner), pretreated with SCFAs for 1 h, and stimulated with 1 μg/mL of H37Rv lysate or 10 ng/mL LPS (Sigma-Aldrich, E. coli serotype 055:B5). Cells were incubated for 24 h or 7 days at 37°C in a 5% CO2 environment (n = 6 to 11). Alternatively, PBMCs were pretreated for 1 hour (37°C, 5% CO2) with ranolazine (ITK Diagnostics), trimetazidine (Sigma), pertussis toxin (Enzo Life Sciences), etomoxir (Sigma) (inhibitors of β-oxidation, n = 3), aspirin (Aspégic injection powder, n = 3), cycloheximide (Sigma, n = 6 to 7), anti-IL-10 antibody IgG2a (BioLegend, n = 10 to 12), or IgG2a isotype control (BioLegend, n = 10 to 12) prior to stimulation. Cell culture supernatants were collected and stored at −20°C for cytokine measurements, performed by ELISA: TNF-α, IL-1β, IL-17A, IL-22, and IL-1Ra (R&D Systems) and IL-6, IFN-γ, and IL-10 (Sanquin).
7
Flow Cytometric Analysis of HLA and Cell Cycle
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Surface expression of HLA-A/B/C (Biolegend, #311434) on tumor cells was investigated using AlexaFluor-647-labeled antibodies and flow cytometry. A corresponding IgG2a isotype control was used (Biolegend, #400203). Cell cycle analysis was performed by staining nuclei with DAPI (1 ng/μL) for at least 4 h. DNA fragmentation (subG1) was determined by analyzing the DNA content/distribution according to a well-established protocol [46 (link)].
8
Immunofluorescence Staining of Pyroptotic BMDMs
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Pyroptotic BMDMs were fixed for 1 hour at room temperature (RT) in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, 15710), then incubated with PBS containing 50 mM NH 4 Cl (Sigma, A9434) for 10 min and washed in the same buffer. Samples were incubated in PBS containing 10 µg/mL of anti-mouse CD68 (Biolegend, 137001) or IgG2a isotype control (Biolegend, 400501) and 0.25% of gelatin for 1 hour at RT. After a washing step, samples were incubated in a solution containing 10 µg/mL of anti-Rat FITC conjugated antibody (Jackson ImmunoResearch, 712-096-153) and 0.25% of gelatin for 1 hour at RT. Finally, samples were washed in PBS, incubated in PBS containing 10 µg/mL of Hoechst 33342 (Invitrogen, H3570) for 10 min at RT, and coverslips were mounted in Mowiol following further washing steps in PBS and distilled water. Images were acquired using the UltraView VOX microscope and data were analysed using the FIJI software.
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