Splenocyte culture was performed as previously described (28 (link)). Briefly, spleens from control and infected mice were removed aseptically and pressed through a sterile fine-wire mesh with 10 ml RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 25 mM Hepes, 0.12% gentamicin and 2 mM glutamine. Cell suspensions were collected by centrifuging at 350 ×g for 10 min. Erythrocytes were lysed with cold 0.17 M NH4Cl and cells were washed twice with fresh medium. Spleen cell viability was determined by trypan blue exclusion and was >90%. Spleen cells were adjusted to a final concentration of 107 cells/ml in RPMI-1640 supplemented with 10% heat-inactivated FCS. Aliquots of 5×106 cells/well were incubated in 24-well flat-bottom tissue culture plates (Falcon) in triplicate for 48 h at 37°C in a humidified 5% CO2 incubator. Cytokines (IFN-γ, TNF and IL-10) in the culture supernatant and plasma samples were measured by enzyme-linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA).
24 well flat bottom tissue culture plates
Manufactured by BD
Sourced in United States
The 24-well flat-bottom tissue culture plates are a laboratory equipment used for cell culture applications. These plates provide a standardized and consistent platform for growing and maintaining cells in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Four well chamber slide, by Thermo Fisher Scientific (1 mentions)
T75 tissue culture flasks, by Sarstedt (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Optiprep, by Axis-Shield (1 mentions)
Galaxy 170r, by Eppendorf (1 mentions)
Tri carb 2910tr liquid scintillation counter, by PerkinElmer (1 mentions)
Ultima gold, by PerkinElmer (1 mentions)
7 protocols using 24 well flat bottom tissue culture plates
1
Splenocyte Culture and Cytokine Measurement
2
Quantification of Cytokine Secretion
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For the quantification of cytokines, splenocytes were harvested from mice at the indicated time points (day 0, 3 and 5) [37 (link)]. Spleen cells were adjusted to a final concentration of 107 cells/ml in RPMI-1640 supplemented with 10% heat-inactivated FCS. Aliquots (500 µl/well) of the cell suspensions were incubated in 24-well flat-bottom tissue culture plates (Falcon®) in triplicate for 48 h at 37 °C in a humidified 5% CO2 incubator. Supernatant fractions were collected and stored at − 80 °C for use in the assays for detecting cytokines.
Transforming growth factor beta (TGF-β) expression was measured using commercial enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's protocol (R&D Systems, Inc. Minneapolis, MN, USA). The OD values were measured using a microplate reader at 450 nm. The cytokine concentration of each sample was calculated using a standard curve generated using recombinant cytokines.
Transforming growth factor beta (TGF-β) expression was measured using commercial enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer's protocol (R&D Systems, Inc. Minneapolis, MN, USA). The OD values were measured using a microplate reader at 450 nm. The cytokine concentration of each sample was calculated using a standard curve generated using recombinant cytokines.
3
Cytokine Profiling of Splenocytes
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Mice were killed at the indicated time points (day 0, 3 and 5) and the splenocytes were havested in order to detect the levels of cytokines [16 (link)]. Concentration of spleen cells was adjusted to 107 cells/ml using RPMI-1640 supplemented with 10% heat-inactivated FCS.. Aliquots (500 μl/well) of the cell suspensions were incubated in 24-well flat-bottom tissue culture plates (Falcon®) in triplicate for 48 h at 37 °C in a humidified 5% CO2 incubator. Following the supernatant fractions were collected and stored at − 80 °C until the detection of cytokines.
Commercial enzyme linked immunosorbent assay (ELISA) detected levels of IFN-γ and IL-10 in the supernatant samples (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The results were calculated by a standard curve generated using recombinant cytokines after obtaining the OD values.
Commercial enzyme linked immunosorbent assay (ELISA) detected levels of IFN-γ and IL-10 in the supernatant samples (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The results were calculated by a standard curve generated using recombinant cytokines after obtaining the OD values.
4
Splenocyte Isolation and Culture Protocol
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Splenocyte culture was performed as previously described (Wang et al. 2009) . Briefly, the spleens from normal and infected mice were removed aseptically and pressed through a sterile fine-wire mesh with 10 mL of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mM Hepes, 0.12% gentamicin, and 2 mM glutamine. The resulting cell suspensions were pelleted by centrifuging at 350 g for 10 min. The erythrocytes in the splenocyte suspension were lysed with cold 0.17 M NH 4 Cl and the remaining cells were washed twice with fresh medium. After the erythrocytes had been lysed, the splenocytes were cultured (500 μL/well) into 24 -well flatbottom tissue culture plates (Falcon) in triplicate and incubated for 48 h at 37°C in a humidified 5% CO 2 incubator. The supernatants were collected and stored at -80°C until they were used to detect cytokines and NO levels.
5
Splenocyte Cytokine Profiling Assay
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Splenocytes were harvested and cultured essentially as previously described (Chen et al. 2009) . Aliquots (5 × 10 6 /well) of the cell suspensions were seeded in 24-well flat-bottom tissue culture plates (Falcon) in triplicate for 48 h at 37 °C in a humidified 5% CO 2 incubator. Supernatant fractions were collected and stored at -80 °C until they were assayed. Levels of IFN-γ, TNF-α and IL-10 in culture supernatants were measured using enzyme linked immunosorbent assays (ELISA) (R&D Systems, Minneapolis, MN, USA).
6
Isolation of Rabbit PBMCs Using OptiPrep
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Fresh blood collected from female New Zealand white rabbits (Covance Research Products, Denver, PA, USA) was used in this study. Peripheral blood mononuclear cells (PBMCs) including monocytes were isolated using OptiPrep (Axis‐Shield, Oslo, Norway) according to a modified manufacturer's protocol for rabbit blood. Twofold diluted rabbit's blood was layered on OptiPrep density solution. After density gradient centrifugation at 700 g for 20 min with nonaccelerator and nonbrake modes, isolated PBMCs were washed two times with phosphate‐buffered saline (PBS; GE Healthcare Life Sciences Hyclone Laboratories, Logan, UT, USA) by centrifugation at 250 g for 10 min. Cells were resuspended in RPMI‐1640 medium (Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences Hyclone Laboratories) and 1% penicillin/streptomycin (Life Technologies) at a concentration of 2 × 106 cells·mL−1. Subsequently, cell suspensions were transferred to T75 tissue culture flasks (Sarstedt, Newton, NC, USA), four‐well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA), or 24‐well flat bottom tissue culture plates (Becton Dickinson, Flanklin Lakes, NJ, USA). After incubation for 2 h at 37 °C in humidified 5% CO2 in a gas incubator (Galaxy 170R; Eppendorf, Enfield, CT, USA), nonadherent cells were removed by washing with PBS 15 .
7
Fluciclovine Uptake Kinetics in Cells
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Uptake experiments were performed as described [28] with minor modifications. Cells (5×10 4 per well) were suspended in medium and seeded in 24-well flat-bottom tissue culture plates (Becton Dickinson, East Rutherford, NJ). After 3 days, cells were washed twice with sodium buffer (140 mM NaCl, 5 mM KCl, 5.6 mM D-glucose, 0.9 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES, pH 7.3 at 37 °C) or choline buffer (sodium chloride was replaced with an equivalent concentration of choline chloride), and then incubated with [ 14 C]fluciclovine at a final concentration of 10 μM for 5, 15, 30, and 60 min at 37 °C in air. Uptake was stopped by removing the tracer solution and rapidly washing the cells twice with ice-cold buffer. The cells were lysed in 0.1 N NaOH, and radioactivity was measured with a Tri-Carb 2910TR liquid scintillation counter (PerkinElmer, Waltham, MA) and Ultima Gold (PerkinElmer). The protein concentration of the cell lysate was determined with a VersaMax microplate reader (Nihon Molecular Device K.K., Osaka, Japan) using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Tracer uptake is expressed as nmol/mg protein.
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