The following fluorophore conjugated monoclonal antibodies were used: CD3 (clone SP34-2), CD4 (clone L200), CD95 (clone DX2), Ki67 (clone B56), CD16 (clone 3G8), γδ TCR (clone B1), IFN-© (clone B27), TNFα (clone Mab11) (BD Biosciences), Perforin (clone Pf-344) (Mabtech), CD107a (clone eBioH4A3), CD28 (clone CD28.2); CD8 (clone 3B5) and Granzyme B (clone GB12; Life Technologies). Briefly, lymphocyte single cell suspensions were washed with PBS supplemented with 0.2% heat-inactivated human serum (Sigma), and incubated with different cocktails of fluorophore-labelled monoclonal antibodies during 20 minutes at room temperature [73 (link)], fixed and permeabilized using the FoxP3 permeabilization reagent (eBioscience). After 30 minutes incubation at 4°C, the cells were washed with FoxP3 washing buffer and intracellularly stained with Ki67 and GrzB for 20 minutes. The cells were washed and resuspended in PBS for flow cytometry analysis. For tetramer staining using samples from MamuA*01+ macaques, the CM9 tetramer was added to the samples 5 minutes prior to the addition of the antibody cocktail for surface staining [73 (link)]. The samples were acquired on a Fortessa or LSRII flow cytometer (BD Biosciences, San Jose, CA) and the data were analyzed using the FlowJo software platform (Tree Star, Inc., Ashland, OR).
Heat inactivated human serum
Manufactured by Merck Group
Sourced in United States, United Kingdom
Heat-inactivated human serum is a laboratory product derived from human blood. It is processed through a heat-inactivation procedure to inactivate any potential pathogens. The product serves as a component in various cell culture and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Endotoxin free bsa, by Merck Group (3 mentions)
Il 1β, by Merck Group (3 mentions)
Dmem f12 medium, by Lonza (2 mentions)
Cd16 clone 3g8, by BD (1 mentions)
Cd3 clone sp34 2, by BD (1 mentions)
Cd4 clone l200, by BD (1 mentions)
Tnf α clone mab11, by BD (1 mentions)
Perforin clone pf 344, by Mabtech (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Ifn gamma, by BioLegend (1 mentions)
Dmem f12, by Lonza (1 mentions)
X vivo 10 media, by Lonza (1 mentions)
15 epi lipoxin a4, by Cayman Chemical (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
20 protocols using heat inactivated human serum
1
Multiparameter Flow Cytometry for Cellular Phenotyping
2
Isolation and Differentiation of Human Macrophages
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Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated from peripheral blood. The samples were centrifuged at 400 g for 10 min at room temperature (RT), the plasma was discarded, and the cellular portion was diluted in phosphate-buffered saline (PBS). PBMC were isolated by density–gradient centrifugation using Histopaque-1077 (Sigma-Aldrich). The collected PBMC fraction was treated with ACK lysis buffer to lyse erythrocytes and washed twice with PBS. For macrophages differentiation, monocytes were cultivated for 7 days in RPMI 1640 media supplemented with penicillin (10,000 U/mL, GIBCO), streptomycin (10,000 μg/mL, GIBCO), 1% glutamine (GIBCO), and 10% heat-inactivated human serum (Sigma Aldrich) at 37°C in a 5% CO2 atmosphere.
3
Tendon Cell Inflammatory Response Assay
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Tendon-derived stromal cells isolated from healthy hamstring and diseased supraspinatus were seeded at a density of 30,000 cells per well in a 12-well plate (mRNA) or 60,000 cells in a 6-well plate (flow cytometry). Cells were allowed to reach 80% confluence prior to stimulation with IL-1β (10 ng/mL-1). Tendon cells were incubated in DMEM F12 medium (Lonza) containing 1% heat-inactivated human serum (Sigma). Medium containing sterile filtered 0.1% endotoxin-free BSA (Sigma) diluted in PBS was used for vehicle-only controls. After IL-1β or vehicle treatment, cells were incubated for 24 hours at 37 °C and 5% CO2 until harvest of the lysate for mRNA or flow cytometry.
4
Cytokine-induced Expression of Inflammatory Genes in Tendon Cells
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IL-1β and IFNγ respectively induce NF-κB and IFN target genes expressed in diseased shoulder tendon tissues8 11 (link) and were also identified in diseased Achilles tendon tissues in the current study. We therefore investigated if treatment of cultured tendon cells with these cytokines induced more profound expression of SFA markers and NF-κB and IFN target genes in diseased relative to healthy tendon-derived cells. Tendon-derived cells were isolated from healthy hamstring and diseased Achilles tendons using previously described protocols11 (link); passage 1–3 cells were used for all experiments. Cells were grown until 80% confluence prior to stimulation with IL-1β (10 ng/mL, Sigma) or IFN gamma (20 ng/mL, BioLegend) in medium (DMEM F12, Lonza) containing 1% heat-inactivated human serum (Sigma). Non-treated (vehicle only) cells served as experimental controls. After cytokine/vehicle treatment, cells were incubated at 37°C and 5% CO2 for 24 hours until experimental harvest for mRNA or flow cytometry.
5
Tendon Stromal Cell Response to LPS and Lipoxin A4
6
Infection and Activation of PBMCs by Trypanosoma Antigens
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Heparinized peripheral blood samples were collected and PBMCs were obtained (13 (link), 18 (link)). PBMCs, resuspended at a concentration of 107 cells/ml, were infected using a ratio of 10 parasites/cell, incubated at 37°C in 5% CO2 for 3 h, and washed for the removal of free TRPs. RPMI supplemented with 5% heat-inactivated human serum (Sigma-Aldrich, St. Louis, MO, USA), antibiotics (penicillin, 200 U/ml; and streptomycin, 0.1 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA), and L-glutamine (1 mM) (Sigma-Aldrich, St. Louis, MO, USA) was added to a final volume of 1 ml. Infected PBMCs were reincubated at 37°C in 5% CO2 for 14 h. Brefeldin A (1 μg/ml, Sigma-Aldrich, St. Louis, MO, USA) was added, and cultures were reincubated for an additional 4 h. Non-stimulated controls (MED) were included.
For the blocking experiments, 3 × 105 PBMCs were incubated with anti-CD1d antibodies (25 μg/ml) and 20 μg/ml of TRP antigens (TRP-SA). Cultures were performed as described above and included PBMCs incubated with media only, media plus anti-CD1d, TRP-SA only, and TRP-SA plus anti-CD1d.
For the blocking experiments, 3 × 105 PBMCs were incubated with anti-CD1d antibodies (25 μg/ml) and 20 μg/ml of TRP antigens (TRP-SA). Cultures were performed as described above and included PBMCs incubated with media only, media plus anti-CD1d, TRP-SA only, and TRP-SA plus anti-CD1d.
7
ADCC Evaluation of BCA101 in EGFR-Expressing Cells
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ADCC of BCA101 was assessed using TGFβ pretreated NK cells and A431 (EGFRhigh) or MCF7 (EGFRlow) cell lines, procured from ATCC. NK cells were isolated from frozen or fresh human peripheral blood mononuclear cells (PBMC) using Human NK-cell negative selection kit (Stemcell Technologies Inc.) and EasySep magnet (Stemcell Technologies Inc.). About 1 million enriched NK cells were left untreated or cultured with 1 ng/mL of TGFβ (BPS Biosciences), TGFβ plus cetuximab or BCA101 (56 nmol/L) for 72 hours in a 12-well plate in NK MACS (Miltenyi Biotec) media containing 1% pen strep (GIBCO, Thermo Fisher Scientific), 1% heat-inactivated human serum (Sigma-Aldrich), and 1% NK supplement (Miltenyi Biotec). These pretreated NK cells were used as effectors in ADCC assay. BCA101 (56 nmol/L) was added to target tumor cells and preincubated for 1–2 hours, after which, pretreated NK cells were added at a ratio of 1:5 (5,000 target cells: 25,000 NK cells) in a 96-well plate. The assay plates were incubated for around 24 hours. Cytotoxicity was measured by evaluating protease release using CytoToxGlo (Promega). Luminescence readout was taken using a plate reader (Cytation5 BioTek plate reader) and data were analyzed using GraphPad Prism software (version9). Data are plotted as percentage change in cytotoxicity over human IgG control group.
8
Resetting PBMC for TGN1412/TAB08 Evaluation
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Cells were cultured in RPMI 1640 supplemented with l -glutamine (Gibco), non-essential amino acids (Gibco), HEPES (Applichem), β-mercaptoethanol (Gibco), sodium pyruvate (Gibco), penicillin/streptomycin, and 10% AB-positive heat-inactivated human serum (Sigma) (AB medium). PBMC were first cultured for 2 days at a high cell density (1 × 107/ml) in AB medium to reset them to tissue-like conditions without changing their cellular composition, thereby allowing reactivity to TGN1412/TAB08 in the secondary cultures. For these, cells were harvested and cultured under standard conditions (1 × 106/ml) in 48-well flat-bottom tissue culture plates in a final volume of 0.6 ml of supplemented RPMI/AB medium for 5 days in a humidified incubator at 37°C with 5% CO2. GMP-grade TAB08 was provided by TheraMAB GmbH. Pan HLA II-specific Tü39 antibodies were provided by Hans-Georg Rammensee from Tübingen, Germany. Isotype control IgG2a was from Abcam. Fab fragments were prepared using the Pierce Fab Preparation Kit (Thermo Scientific).
9
Macrophage Phagocytosis and Intracellular Trafficking
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Macrophage colony-stimulating factor (MCSF; ImmunoTools), phorbol 12-myristate 13-acetate (PMA; Sigma), LysoTracker Red DND-99 (Molecular Probes), PI (Life Technologies), heat-inactivated human serum (Sigma), and Accutase (Sigma) were used. The primary antibodies were a goat anti-group A polysaccharide antibody (Abcam), a mouse anti-LAMP1 antibody (BD Bioscience), a mouse anti-ubiquitin antibody (FK2; Enzo), and a mouse anti-SQSTM1 (p62) antibody (Abcam). The secondary antibody was an Alexa Fluor-conjugated donkey anti-mouse or donkey anti-goat antibody (Invitrogen).
10
Serum-Supplemented Saliva Growth Media
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Heat inactivated human serum (Sigma, United States) was added to Sterile pooled human saliva (SPHS) prepared as described above at final concentrations of 1%, 2%, 3.5%, and 5% (v/v) to obtain a total of four different growth media.
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