After treatment, EAU was scored every 3 to 4 days per week until the mice treated with α-MSH had resolved the inflammation. The eyes were enucleated for preparation of RPE eyecups. The RPE eyecups were made based on our previously published method.5 (link), 7 (link), 11 (link), 39 (link) Eyes were placed in ice-cold 0.01M PBS (Lonza) and the connective tissue and optic nerve were removed from the eyeball. A circumferential cut was made around the limbus to remove the entire anterior segment including the cornea, iris, ciliary body and lens. The neural retina was gently removed from the RPE monolayer and discarded. The remaining posterior of the eye (RPE eyecups) contain only the RPE monolayer, choroid and the sclera. The RPE eyecup was placed into the well of a 96-well round bottom tissue culture plate (Corning, Corning, NY, USA) containing 200uL Serum-free Media (SFM): RPMI 1640 supplemented with a 1/500 dilution of ITS+ (Insulin/Transferrin/Selenium + linoleic acid) (Sigma-Aldrich, St. Louis, MO), and 0.1% sterile BSA solution (Sigma-Aldrich). The cultures were incubated for 24 hours at 37C in 5% CO2, and the conditioned media (CM) was collected, centrifuged at 2100g for 5 minutes, and the supernatant was used immediately in the assays as RPE eyecup CM.
96 well round bottom tissue culture plate
Manufactured by Corning
Sourced in United States
The 96-well round bottom tissue culture plate is a laboratory equipment used for various cell culture applications. It provides a standardized platform with a grid of 96 individual wells, each with a round bottom shape. The plate is designed to facilitate the growth, maintenance, and analysis of cells in a controlled and organized manner.
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Lab products found in correlation
Rpmi 1640, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Mouse il 6 elisa max standard set, by BioLegend (1 mentions)
Dnp has, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Roche (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Lonza (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Glass fiber filter, by PerkinElmer (1 mentions)
Sample bag, by PerkinElmer (1 mentions)
Methyl 3h thymidine, by PerkinElmer (1 mentions)
Bsa fraction 5, by Thermo Fisher Scientific (1 mentions)
Polystyrene round bottom tube, by Corning (1 mentions)
Cellquest software version 3, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
6 protocols using 96 well round bottom tissue culture plate
1
Isolating RPE Eyecups from Mouse Eyes
2
Metabolite Analysis of IgE/Antigen-Activated Mast Cells
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For analysis of metabolites in the supernatant of IgE/antigen-activated mouse BMCMCs, cells were sensitized (or not) overnight with 1 μg/ml dinitrophenyl (DNP)-specific IgE (kindly provided by Dr. Fu-Tong Liu (University of California-Davis) (Liu et al., 1980 (link)). Next day, cells were collected and washed twice in RPMI 1640 without phenol red (Gibco) containing 1 mg/ml DTT (Roche; RPMI DTT). Cells were resuspended at 2× 106 cells/ml in RPMI DTT and seeded at 50 μl aliquots (quadruplicates) in a 96-well round bottom tissue culture plate (Corning). After addition of 50 μl of 20 ng dinitrophenyl30–40-conjugated human serum albumin (DNP-HAS; Sigma)/ml in RPMI/DTT, cells were incubated for 1 hour at 37°C. Cells were then transferred to Bio-pur 1.5 ml microcentrifuge tubes (Eppendorf) and centrifuged for 5 min at 500 x g, 4°C. After transfer of supernatant to fresh tubes, supernatant and pellets were frozen in liquid N2 and stored at −80°C. IL-6 was measured using the ELISA MAX Standard Set Mouse IL-6 (Biolegend).
For assessment of Substance P-mediated mast cell activation and mediator release, ePMCs or hu PBCMCs (2.5× 105 cells in 50 μl RPMI DTT) were either pre-treated with 100 μM QWF or DMSO for 10 minutes, followed by stimulation with 10 μM or 100 μM Substance P, respectively. After 1 hour incubation, cells were processed as described above for BMCMCs.
For assessment of Substance P-mediated mast cell activation and mediator release, ePMCs or hu PBCMCs (2.5× 105 cells in 50 μl RPMI DTT) were either pre-treated with 100 μM QWF or DMSO for 10 minutes, followed by stimulation with 10 μM or 100 μM Substance P, respectively. After 1 hour incubation, cells were processed as described above for BMCMCs.
3
Preparation of RPE Eyecup Conditioned Media
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The RPE eyecups were made as previously reported [19 (link)]. The enucleated eyes were dissected, making RPE eyecups consisting of the RPE monolayer with underlying choroid and sclera with the neuroretina removed. The RPE eyecups were placed into the wells of a 96-well round bottom tissue culture plate (Corning, Corning, NY, USA) containing 200 µL of serum-free media (SFM) (RPMI 1640 supplemented with 10 mM HEPES, 1 mM sodium pyruvate, and nonessential amino acids (Lonza), along with 0.2% ITS+1, 0.1% BSA, and 10 µg/mL gentamycin (Sigma-Aldrich). The cultures were incubated for 24 h at 37 °C in 5% CO2. The conditioned media (CM) was collected, centrifuged, and the supernatants were used as RPE eyecup-CM.
4
Measuring PBMC Proliferation Modulation
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PBMC (106/mL final concentration) were seeded in a 96-well round bottom tissue culture plate (Corning, Amsterdam, The Netherlands), activated using anti-CD3 and anti-CD28 monoclonal antibody, as described earlier, and cultured with different conditioned media or isolated extracellular vesicles in order to explore their effects on the activity of PBMC. Proliferation was measured after the addition of [methyl-3H]-thymidine (1 Ci/well; PerkinElmer, Courtaboeuf, France) for the last 18 h of each time point. At the end of each time point, cultured cells were harvested on a glass fiber filter (PerkinElmer, Courtaboeuf, France) using a Tomtec harvester (Wallac, Turku, Finland). The filter was then sealed in a sample bag (PerkinElmer, Courtaboeuf, France) after drying and addition of scintillation liquid (Beckman Coulter, Brea, CA, USA). Radioactive thymidine, incorporated into replicated cellular DNA by proliferative cells, was detected by scintillation counting using a 1450 Trilux β-counter (Wallac, Turku, Finland). Each proliferation assay was carried out in triplicate and estimated in count per minute (cpm) or, when stated, the results were normalized compared with non-treatment conditions to obtain the relative proliferation. All data are presented as the mean values and standard error of at least three independent experiments.
5
Cytotoxicity Assay with Effector-Target Cells
6
CBMC Proliferation Assay with Immune Stimulants
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