Chemical reagents, culture media, solvents and positive controls were purchased from Sigma-Aldrich (Darmstadt, Germany).
Positive control
Manufactured by Merck Group
Sourced in Germany, United States
A positive control is a sample that is known to produce a positive result in a test or assay. It is used to verify the proper functioning of the test system and to ensure the validity of the test results. Positive controls are an essential component in various laboratory procedures and are typically included in test kits or protocols to demonstrate that the test is performing as expected.
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7 protocols using positive control
1
Reagents and Controls for Assays
2
Antimicrobial Activity of Plant Extract
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The extract (at concentrations from 5 to 0.005 mg/mL) was tested against Bacillus cereus (food isolate), Staphylococcus aureus (ATCC 11,632), Listeria monocytogenes (NCTC 7973), and Enterococcus faecalis (ATCC 19,433) (Gram-positive bacteria) and Escherichia coli (ATCC 35, 218) and Salmonella typhimurium (ATCC 13,311) (Gram-negative bacteria) (Soković et al., 2010) . The fungi Aspergillus fumigatus (ATCC 1022), Aspergillus ochraceus (ATCC 12,066), Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Penicillium funiculosum (ATCC 36,839), and Penicillium verrucosum var. cyclopium (food isolate) were also tested (Soković and van Griensven, 2006) . The procedures and the origin of the microorganisms were previously described by the authors. As positive controls (Sigma-Aldrich), streptomycin and ampicillin were used for the antibacterial activity, while ketoconazole and bifonazole were used for the antifungal activity. The results were given as minimum inhibitory (MIC) and minimum bactericidal (MBC) or minimum fungicidal (MFC) concentrations (mg/mL).
3
Peripheral Blood Mtb Antigen Stimulation
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Peripheral whole blood samples were collected in heparinized tubes for all the subjects in the biomarker screening, biomarker validation and clinical validation groups. Whole blood samples from the subjects were incubated with the Mtb antigens ESAT-6, CFP-10 (each at a concentration of 10 µg/mL; Sangon Peptide Technologies, Shanghai, China), 6-phosphonohexanoic acid (5 µg/mL; positive control; Sigma-Aldrich, St. Louis, MO), or without stimulants (negative controls). Following incubation at 37 °C for 20–24 h, supernatants were harvested and cryopreserved at − 80 °C for batched analysis.
4
Cytokine Estimation and TLR Interaction
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Estimation of cytokines and identification of TLR interaction study was designed and performed based on previously used method from our lab (35 (link)). These TLR knockout cell lines were obtained from BEI Resources established by the National Institute of Allergy and Infectious Diseases (NIAID) Maryland, USA. RAW264.7, ΔTLR4 and ΔTLR2/4 mouse macrophages were seeded (0.3×106 cells per well) in 24 well tissue culture plates and incubated for 4 hours at 37° C for adherence. Then cells were treated with different concentrations of protein (1μg/ml, 2μg/ml and 4μg/ml) or lipopolysaccharide (LPS) (50ng/ml; Positive control) (Sigma USA) along with Control (no protein). After 48 hours of treatment, supernatants were collected and cytokines were estimated in culture supernatants. The estimation of pro-inflammatory cytokines such as TNFα, IL-12 and IL-6, and anti-inflammatory cytokine IL-10 was performed using mouse ELISA kit following the manufacturer’s instructions.
5
Calreticulin Upregulation Kinetics
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5 × 105 cells were seeded in a T25 cm2 flask with 5 mL DMEM. To determine the kinetics of calreticulin upregulation, ID8 cells were treated with 1 Gy radiation or doxorubicin (positive control, 25 μmol/L; Sigma) and harvested 4, 6, 12, and 24 hours after exposure as previously described, washed twice with cold PBS, followed by staining with a calreticulin-specific antibody (Abcam; catalog no. ab83220, RRID:AB_1859755), Annexin V (Thermo Fisher Scientific; catalog no. 88-8007-72, RRID:AB_2575165), which recognizes phosphatidylserine on the surface of apoptotic cells, plus vital dye 4,6-diamidino-2-phenylindole (DAPI), which stains dead cells. Isotype-matched IgG antibody was used as a negative control (Abcam; catalog no. ab91357, RRID:AB_2888649), and the analysis was limited to living (DAPI-negative) tumor cells (104 (link)).
6
Staphylococcal Enterotoxin B ELISPOT Assay
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Staphylococcal enterotoxin B (SEB) was used as a positive control (Sigma-Aldrich), and either cells alone (negative control, no peptides) or actin (JPT Technologies) was used as an irrelevant antigen control. HST cells were plated at 105/well on anti–IFN-γ–coated ELISPOT plates (MilliporeSigma). Positive responses were defined as having more than four times the spot-forming cells obtained in the negative control actin. For epitope mapping, peptides were mixed into pools of 10–15 peptides before T cell stimulation on an ELISPOT. Using matrices, cross-reactive pools were analyzed for common epitopes, and these peptides were then individually tested on ELISPOT to confirm specificity.
7
Macrophage Nitric Oxide Assay with Nanoparticles
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Murine RAW 264.7 macrophages (ATCC)
were grown following standard cell culture practices, as previously
described.26 (link) The day before the experiment,
3.3 × 106 cells/well were plated in a 6-well plate.
The following day, cells were exposed to 0.2 μg/mL LPS (positive
control; Sigma-Aldrich; E. coli 055:B5) or different
concentrations of CDs in fresh media (negative control, 0 μg/mL
CD). After 24 h, 50 μL of supernatant was analyzed for nitric
oxide (NO) production measured as released nitrate (μM) using
Greiss reagent as previously described.26 (link) To determine whether cells uptake CDs, murine RAW 264.7 macrophages
were incubated and grown on a coverslip overnight and then incubated
for 6 h with different CD conjugates at 20 μg/mL before imaging.
were grown following standard cell culture practices, as previously
described.26 (link) The day before the experiment,
3.3 × 106 cells/well were plated in a 6-well plate.
The following day, cells were exposed to 0.2 μg/mL LPS (positive
control; Sigma-Aldrich; E. coli 055:B5) or different
concentrations of CDs in fresh media (negative control, 0 μg/mL
CD). After 24 h, 50 μL of supernatant was analyzed for nitric
oxide (NO) production measured as released nitrate (μM) using
Greiss reagent as previously described.26 (link) To determine whether cells uptake CDs, murine RAW 264.7 macrophages
were incubated and grown on a coverslip overnight and then incubated
for 6 h with different CD conjugates at 20 μg/mL before imaging.
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