Neutrophils were purified from freshly-obtained blood of healthy human donors using the MACSxpress magnetic purification system (Miltenyi Biotechnology, cat. No. 130-104-434), as per manufacturer’s instructions. Viable cells of pneumococcal strain M117-14 were pre-mixed with a capsule-specific hmAb (0.4 μg/mL) and 10% v/v baby rabbit complement (Bio-Rad), and the mixture incubated for 30 minutes at 37°C, with shaking at 150 rpm. Approximately 4 x 103 colony-forming units (CFU) from this mixture were then transferred to wells of a clean microtitre plate. To each well containing the pneumococci-hmAb mixture, 4 x 105 neutrophils were added (MOI of 1:100). The plate was subsequently incubated for 45 minutes at 37°C, with shaking at 150 rpm. The numbers of surviving bacteria were calculated by plating serial dilutions, and the results were expressed as a percentage of the inoculum CFU.
Baby rabbit complement
Manufactured by Bio-Rad
Sourced in United States
Baby rabbit complement is a laboratory reagent used in various immunological and biochemical assays. It contains the components of the complement system obtained from the serum of baby rabbits. The complement system is a group of proteins that play a crucial role in the body's immune response. This product can be used to study and evaluate the activation and function of the complement system in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
7 amino actinomycin 7 aad, by BD (2 mentions)
Polymorphprep, by Axis-Shield (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Raw264.7 macrophage, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Penicillin streptomycin solution, by American Type Culture Collection (1 mentions)
7 protocols using baby rabbit complement
1
Neutrophil-Mediated Pneumococcal Killing
2
Neutrophil-mediated Streptococcus pyogenes killing
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Neutrophils were isolated from blood drawn from healthy donors using PolymorphPrep (Axis-Shield), and adjusted to a final concentration of 2 × 106 cells/ml. Mid-log phase GAS (2 × 105) were incubated with 50 μl of PrsA antisera or naïve rabbit sera (pooled sera from two rabbits before PrsA immunization) for 30 min at 37 °C. Neutrophils were preincubated with 4% baby rabbit complement (Bio-Rad, Cat. No. C12CA) for 10 min and added to bacteria at a MOI of 1, briefly centrifuged to ensure contact, and incubated for 30 min at 37 °C. After incubation, samples were serially diluted in sterile water to lyse cells and plated onto THY agar. Sera from pre-immunized rabbits were used as control for the baseline bacterial killing of neutrophils. At minimum, each serum or serum combination was tested in triplicates with 2–3 independent experiments to ensure statistical confidence. Bacterial survival was calculated by dividing the number of surviving bacterial CFUs in PrsA antisera-treated group by the number of bacterial CFUs recovered from the naïve sera-treated group.
3
BTLA Antibody Depletion Assay
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Example 14
Splenocytes from humanised mice were incubated with 10% baby rabbit complement (BioRad) and anti-BTLA antibodies (or an isotype control or a positive control depleting anti-CD20 antibody; clone SA271G2 from Biolegend) at 20 μg/ml for 15 min at 37° C. Whilst anti-CD20 antibody depleted the majority of B220+ B cells, anti-BTLA antibodies did not deplete either B220+ or CD4+ cells (
4
Complement-Mediated Bacteriolysis of A. baumannii
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Complement-mediated bacteriolysis was evaluated using A. baumannii clinical isolates, Ab16, Ab29, and Ab35. The A. baumannii clinical isolates were cultured as previously described (Method 2.7). Mice sera were heat-treated for 30 min at 56 °C to inactivate the complement. The heat-treated pooled mice sera (5%) were mixed with a 5% baby rabbit complement (Bio-Rad, Hercules, CA, USA). Later, the mice sera and baby rabbit complement mixture were added with the diluted Ab16, Ab29, and Ab35 cultures. The mixture was incubated at 37 °C for 1 h, followed by plating onto MH agar plates. The plates were incubated overnight at 37 °C, and the number of bacterial colonies formed was determined. The percentage of bacteriolysis was calculated as shown below:
5
Opsonization of Pathogenic Bacteria for Macrophage Infection
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RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were routinely grown in complete high glucose Dulbecco’s modified Eagle’s medium (DMEM) (ThermoFisher, Carlsbad, CA, USA) with heat-inactivated fetal bovine serum (Gemini BioProducts, Calabasas, California, USA) and penicillin-streptomycin solution (ATCC, Manassas, VA, USA). Cells were incubated at 37 °C with 5% CO2. For infection, 5 × 104 cells per well were plated in a 96-well plate and were left to adhere for 1 h prior to infection. Bacterial inoculum was used at an MOI of 5 (2.5 × 105 CFU) was incubated in the presence or absence of heat-inactivated immune sera from HKST, OMVs, or HKST + OMVs (final concentration of 10%) with baby rabbit complement (Bio-Rad, Hercules, CA, USA) for 30 min at 37 °C with 5% CO2. Opsonized bacteria were transferred to plate with cells and incubated for 30 min at 37 °C with 5% CO2. After infection, cells were washed, lysed with 0.1% Triton X-100 (MilliporeSigma, Burlington, MA, USA) in PBS, and plated on LB agar for 24 h. Cells were counted after 24 h and values were normalized to unopsonized bacterially infected cells.
6
Antibody-mediated complement-dependent cytotoxicity
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MOLT-4 cells (105 cells in 100 µl) were plated on each well of a 96-well V-bottom plate. The cells were incubated with the indicated concentrations of 92R, 91R (anti-hCCR9) or isotype-matched control mAb (30 min, 37°C), centrifuged, and washed. Active or 56°C heat-inactivated baby rabbit complement (25%; AbD Serotec) was added in serum-free Dulbecco’s modified Eagle’s medium with 1% BSA (1 h, 37°C). The complement in M4 serum was also heat inactivated (56°C, 30 min). The number of non-viable cells was evaluated by flow cytometry after staining the cells with the viability exclusion marker 7-aminoactinomycin (7-AAD; BD Biosciences; 10 min, 4°C); each condition was analyzed in triplicate. Specific lysis was calculated as: 100 × (% dead cells with active complement − % dead cells with inactive complement)/(100% − % dead cells with inactive complement).
7
Complement-Mediated Cytotoxicity Assay
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MOLT-4 cells (105 target cells/100 μl) were plated in a 96-well V-bottom plate, incubated with indicated concentrations of anti-hCCR9 or an isotype-matched control mAb (30 min, 37 °C), centrifuged and washed. Active or 56 °C heat-inactivated baby rabbit complement (25%; AbD Serotec) was added in serum-free Dulbecco's modified Eagle's medium with 1% BSA (1 h, 37 °C). Cells were stained with the viability exclusion marker 7-aminoactinomycin (7-AAD; BD Biosciences; 10 min, 4 °C) and the number of non-viable cells evaluated by flow cytometry; each condition was analyzed in triplicate. Specific lysis was calculated as 100x (% dead cells with active complement - % dead cells with inactive complement)/(100% - % dead cells with inactive complement).
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