Splenocytes were cultured in RPMI (Cellgro/Mediatech) supplemented with 10% FBS at a concentration of 5x105 cells per well in a 96-well round-bottom plate. In vitro stimulation was performed by adding αCD3 (clone 145-2C11) and αCD28 (clone 37.51) (eBioscience, San Diego, CA) in solution to a final concentration of 1 µg/ml and 2 µg/ml, respectively, or utilizing Cell Stimulation cocktail (PMA/Ionomycin) diluted 1:500 (eBioscience). Fc-mediated depletion was investigated by culturing and treating cells as described, followed by the addition of 25 µL of rabbit complement (Cedarlane Laboratories) for the final 30 minutes of culture at 37°C. At the end of the culture period, cells were washed in FACS buffer prior to fixation using FoxP3/transcription factor kit (eBioscience).
Rabbit complement
Manufactured by Cedarlane
Sourced in Canada, United States
Rabbit complement is a laboratory reagent used in various immunological assays. It serves as a source of complement proteins, which are a critical component of the immune system's complement cascade. Rabbit complement can be used to facilitate the detection and quantification of antibodies, antigens, and other immune complexes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti thy 1.2 at83 a 6, by Cedarlane (4 mentions)
Crystal violet, by Merck Group (2 mentions)
Trypsin collagenase type 1, by Merck Group (2 mentions)
Ara c, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Forskolin fsk, by Merck Group (2 mentions)
αcd3 clone 145 2c11, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor kit, by Thermo Fisher Scientific (1 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Anti cd90.2 53 2 1, by BD (1 mentions)
96 well plate, by Corning (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
Ack lysing buffer, by Quality Biological (1 mentions)
Nk1.1 pk136, by Thermo Fisher Scientific (1 mentions)
Anti thy1.2 30 h12, by Thermo Fisher Scientific (1 mentions)
Facscelesta, by BD (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Foetal bovine serum (fbs), by EQT (1 mentions)
Fetal bovine serum (fbs), by EQT (1 mentions)
29 protocols using rabbit complement
1
In Vitro T Cell Stimulation and Fc-Mediated Depletion
2
HCMV Viral Neutralization Assay
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The viral neutralization assay has been described previously30 . MRC-5 or ARPE-19 cells were seeded on day 1 at 1.2 × 104 or 1.7 × 104 cells/well, respectively, in 50 μl medium per well in 96-well plates. The antibody samples, or the heat-inactivated serum samples, in twofold serial dilutions, were mixed with 6 × 104 pfu/ml of AD169r-GFP virus in equal volume. The mixture was incubated at 37 °C for 1 h and then 50 μl were transferred to MRC-5 or ARPE-19 cells. In the complement-enhanced neutralizing activity assay of gB mAbs, the virus was incubated with the mAbs in the presence or absence of rabbit complement (Cedarlane, Canada) at the final 1:64 dilution. The plates were cultured for 44 h and cells expressing GFP as a surrogate for HCMV infection were then enumerated using Acumen eX3 laser-scanning fluorescence microplate Cytometer (TTP Lab Tech Ltd, United Kingdom). The IC50 values were defined as the concentration of mAb, which achieves a 50% reduction in the number of virus-infected cells. The NT50 titers were defined as the reciprocal serum dilutions that achieved 50% reduction of viral infection. Both IC50 and NT50 values were calculated by four-parameter non-linear curve fittings using GraphPad Prism 8.
3
CDC Activity Assay with Complement
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For CDC activity, target cells (2 × 104 cells) were inoculated into 96‐well plates and incubated with 5% rabbit complement (Cedarlane) and ch6G10A, mu6G10A, control human IgG, or control mouse IgG for 4 hours at 37°C. Cell viability was determined using MTT assay. Assays were done in triplicate at least.
4
Calcein-based Cytotoxicity Assay for D-17 Cells
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The target D-17 cells were incubated with 10 μg/mL calcein AM [25 (link),26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link),35 (link),36 (link),37 (link),38 (link),39 (link),40 (link),41 (link),42 (link)], and were incubated with rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada; final dilution 1:10) in the presence of 100 μg/mL of control dog IgG, E134Bf, H77Bf, and E134Bf-H77scFv. The Calcein release into the medium was determined as indicated in the previous section. Cytolyticity (% lysis) was determined as described previously [27 (link)].
5
Bone Marrow Cell Reconstitution in Mice
6
Antibody-Dependent Enhancement Assay
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ADE activity was measured using semi-adherent K562 cells, as described previously [41 (link)]. Briefly, serially diluted sera samples were mixed with 100 FFU of DENV; these samples were incubated at 37 °C for 2 h in a poly-l -lysine-coated plate in the presence or absence of rabbit complement at a final concentration of 5% (Cedarlane Laboratories, Burlington, Canada) [32 (link)]. Next, 1 × 105 K562 cells were added to the mixture and incubated for 2 days. After immunostaining, viral-infected cells were manually counted. The baseline of the infected cells (without antibody) was log10 2.0 (100 FFU). The infected cell number, which is more than log10 2.5 (i.e., approximately three-fold enhancement from the baseline infected cells), was considered to be ADE. The ADE titer was defined as the maximum serum dilution factor exceeding the ADE threshold.
7
HSV-2 Neutralization Antibody Assay
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In brief, sera samples obtained 21 days after the third vaccination were heat inactivated, serially diluted two-fold (1:4–1:2048) in media containing 10% rabbit complement (Cedarlane, Burlington, NC) and then mixed with HSV-2 (50–100 pfu) and incubated for 1 h at 37 °C42 (link). The samples were then added to Vero monolayer and incubated at 37 °C for 1 h followed by a 1.5% methylcellulose overlay. After 3 days at 37 °C, the overlay was removed and plaques enumerated after staining with Crystal Violet (Sigma-Aldrich, St. Louis, MO). For each sample, the highest dilution producing a ≥50% reduction in plaques was considered the neutralizing antibody endpoint.
8
Splenocyte T-cell Depletion and Activation
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Splenocyte stimulators from 2W-OVA.F1 mice were prepared and their red blood cells were lysed with ACK lysing buffer (Quality biological), followed by 30min incubation with anti-CD90.2 (53–2.1, BD Biosciences) to deplete T cells. Labeled T cells were depleted with two consecutive 35min incubations with rabbit complement (Cedarlane) at 37°C. >40× 106 T-depleted splenocytes were then incubated overnight with 20μg/ml LPS. The following day, 1 × 106 responder cells (Pan-T enriched splenocytes) were plated with 0.5 × 106 stimulators (T-depleted APC’s) in triplicate in a 96-well plate (Corning) and incubated at 37°C overnight. Next, Golgi Plug (BD Biosciences) was added at 1:1000 and incubated for an additional 6h at 37°C. Live/Dead and extracellular staining were performed for 10min and 15min (respectively) on ice, and cells were then fixed with BD Cytofix/Cytoperm according to the manufacturer’s instruction (BD Biosciences). Finally, cells were stained for intracellular IFNg and TNFa and acquired via flow cytometry.
9
Bone Marrow Reconstitution Assay
10
Cytotoxicity Assay for PBMCs
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Briefly, PBMCs (5×105 cells in 250µL FACS buffer) were incubated with 50µL heat-inactivated human serum at 4°C for 1h. After washing with PBS, FACS buffer (200µL) and rabbit complement (50µL, Cedarlane, Hornby, CA, USA) were added (final concentration 20%), and incubation was carried out at 37°C for 30min. After washing with PBS, the cells were incubated in the dark at 4°C for 15min with propidium iodide, and finally 200µL FACS buffer was added. Flow cytometry was carried out using BD FACSCelesta.
Cytotoxicity was calculated, as follows (11 (link)):
where A represented the percentage of dead cells, B was the maximal percentage of dead cells (PBMCs fixed with 70% ethanol), and C was the minimal percentage of dead cells (PBMCs incubated with medium only).
Cytotoxicity was calculated, as follows (11 (link)):
where A represented the percentage of dead cells, B was the maximal percentage of dead cells (PBMCs fixed with 70% ethanol), and C was the minimal percentage of dead cells (PBMCs incubated with medium only).
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