Eight-week-old female BALB/c mice (Charles River Laboratories, Santa Perpetua de Mogoda, Spain) were inoculated with pairwise combinations of the wild type, an opvABON strain, and a ΔopvAB strain at a 1:1 ratio. Bacterial cultures were previously grown overnight at 37°C in LB without shaking. Oral inoculation was performed by feeding the mice with 25 μl of PBS containing 0.1% lactose and 108 bacterial colony-forming units (CFU). Intraperitoneal inoculation was performed with 104 CFU in 200 μl of PBS. Bacteria were recovered from the spleen and the liver of infected mice at 2 days post-infection (intraperitoneal challenge) or 5 days post-infection (oral challenge). A competitive index (CI) was calculated as described elsewhere [48 (link)]. To permit strain discrimination, ATCC 14208 was tagged with trg::MudJ (Kmr), an allele that is neutral for virulence [68 (link)]. When necessary, cross-streaking on green plates with P22 H5 was used to discriminate phage-resistant isolates [65 (link)]. Infection of cultured J774 mouse macrophages, inoculation of guinea pig serum (Sigma-Aldrich), and calculation of competitive indexes in vitro followed previously described protocols [68 (link)]. The Student's t test was used to determine whether the CI's were significant.
Guinea pig serum
Manufactured by Merck Group
Guinea pig serum is a biological material derived from the blood of guinea pigs. It is used as a laboratory reagent in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Bisbenzimide, by Merck Group (2 mentions)
Pronase, by Merck Group (2 mentions)
Anti dinitrophenyl bsa anti dnp, by Merck Group (2 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
2 4 6 trinitrobenzenesulfonic acid, by Merck Group (1 mentions)
Gelatin veronal buffer, by Merck Group (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Prism 5, by GraphPad (1 mentions)
6 protocols using guinea pig serum
1
Competitive Fitness of Salmonella Strains
2
Blastocyst Inner Cell Mass and Trophectoderm Staining
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Allocation of cells to the trophectoderm (TE) and inner cell mass (ICM) in the blastocyst, was performed as previously described [29] (link). Briefly, blastocysts were placed into 0.5% pronase (Sigma Chemical Company, St Louis, MO, U.S.A) at 37°C until the zona pellucida dissolved, before incubation in 10 µM of 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) at 4°C for 10 min. Blastocysts were transferred into 0.1 mg/µl anti-dinitrophenyl- BSA (anti-DNP, Sigma) for 10 min at 37°C and then placed in guinea pig serum (Sigma) with PI (Sigma) for 5 min at 37°C. Finally, embryos were placed in bisbenzimide (Sigma) at 4°C overnight. The following day the embryos were washed through 100% ethanol, mounted in glycerol and visualized using a fluorescence microscope.
3
Blastocyst Cell Lineage Determination
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Allocation of cells to the trophectoderm (TE) and inner cell mass (ICM) in the blastocyst, was performed at 96 h of culture as previously described55 (link). Briefly, blastocysts were placed into 0.5% pronase (Sigma) at 37 °C until the zona pellucida dissolved, before incubation in 10 μM of 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) at 4 °C for 10 minutes. Blastocysts were transferred into 0.1 mg/μl anti-dinitrophenyl- BSA (anti-DNP, Sigma) for 10 minutes at 37 °C and then placed in guinea pig serum (Sigma) with PI (Sigma) for 5 minutes at 37 °C. Finally, embryos were placed in bisbenzimide (Sigma) at 4 °C overnight. The following day the embryos were washed through 100% ethanol, mounted in glycerol and visualized using a fluorescence microscope.
4
Quantitative Complement Deposition Assay
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Immune complexes were formed by adding 10 µL of blue-coupled beads to 96-well u-bottom plates and incubating with 10 µL diluted purified mAb or clinical sample for 2 hours at 37°C and 5% CO2. After incubation, the immune complexes were washed with RPMI medium (Gibco, 21870076). Guinea pig serum (Sigma, 234395) was diluted 1:60 in gelatin veronal buffer (Sigma, G6514), added to the plates, and incubated for 50 minutes at 37°C and 5% CO2 to facilitate binding between complement proteins and anti-RSV F protein immune complexes. The reaction was then stopped by washing the plates twice with 15 mM ethylenediaminetetraacetic acid in PBS. To detect complement deposition, plates were incubated with fluorescein isothiocyanate-conjugated goat anti-guinea pig complement C3 antibody (MP Biomedicals, 0855385) for 20 minutes in the dark. Plates were washed with PBS and fluorescence was measured on a BD LSRFortessa. The results were reported as the average (n = 3 technical replicates) median fluorescent intensity.
5
Cytotoxic Effects of Infliximab and Etanercept on HCC Cells
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For the ADCC assay, human HCC tumor cell lines HepG2 and Hep3B were used as target cells and peritoneal macrophages from BALB/c mice were used as effector cells. Target cells were seeded in a 96-well plate (2,000 cells/well) and pre-incubated with infliximab or etanercept (2, 4, 8, 16, or 32 µg/ml) for 1 h at 37°C. In the control group, infliximab or etanercept was replaced with IgG. Subsequently, the target and effector cells were mixed to a ratio of 1:15 and incubated for 48 h at 37°C. For the CDC assay, target cells were plated in a 96-well plate at a density of 5,000 cells/well. Target cells were then incubated with 5% fresh guinea pig serum (Sigma-Aldrich; Merck KGaA) with active complements and infliximab or etanercept for 6 h at 37°C. Cell viability was determined using the aforementioned CCK-8 assay according to the manufacturer's instructions. Cytotoxicity in each test was calculated as previously described (22 (link)). Briefly, the survival rate of the two treatment groups was calculated use the formula: 100 × (cell concentration of treatment group/cell concentration of the control group). The survival rate of control group was defined as 100% and its inhibition rate was defined as 0%. The curves of the control groups were not illustrated in the figures. The difference in cytotoxicity between infliximab and etanercept treated groups was analyzed by a paired t-test.
6
Borreliacidal Titer Determination
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