The mouse BMDCs were generated as previously described21 . Because our study showed that 12.5 μM of DXM could decrease LPS-induced BMDC function21 , we used 6.25 and 12.5 μM of DXM as the initial concentrations for the following in vitro experiments. BMDCs were pretreated with DXM for 3 h. After incubation, the cells were harvested and washed with PBS. CD4+ T cells were positively enriched from the inguinal LNs of CIA mice (day 28) using MACS cell separation according to the manufacturer’s instruction (Miltenyi Biotec) and cultured with DXM-treated DCs (at DC:T cell = 1:5 ratio) in a 200-ul total volume per condition. In all experiments, in a 96-well flat-bottom plate (Corning), CII (20 ug/ml) or plate-bound anti-CD3 Abs (1 μg/well, Biolegend) were added to the co-culture wells and incubated for 96 h. Supernatants were harvested for measurement of cytokine production, and cell proliferation was detected using [3H] thymidine incorporation with a beta-counter (Beckman Instruments).
Beta counter
Manufactured by Beckman Coulter
Sourced in United States
The Beta counter is a laboratory instrument used for the detection and quantification of beta radiation. It measures the radioactive decay of beta particles emitted from a sample. The core function of the Beta counter is to provide precise and accurate measurements of beta-emitting radionuclides in a variety of research and analytical applications.
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Lab products found in correlation
96 well flat bottom plate, by Corning (2 mentions)
Macs cell separation, by Miltenyi Biotec (1 mentions)
Anti cd3 ab, by BioLegend (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
3h diprenorphine, by PerkinElmer (1 mentions)
Damgo, by Merck Group (1 mentions)
Thymidine, by PerkinElmer (1 mentions)
Methyl 3h thymidine, by PerkinElmer (1 mentions)
Tr22421, by Thermo Fisher Scientific (1 mentions)
Cls9018bc, by Thermo Fisher Scientific (1 mentions)
Phycoerythrin conjugated anti mouse cd4, by BioLegend (1 mentions)
Golgistop, by BD (1 mentions)
Cytofix cytoperm plus kit, by BD (1 mentions)
10 protocols using beta counter
1
Modulation of T Cell Responses by DXM-Treated DCs
2
OVA-specific T cell responses modulated by rHDL
3
Kappa Opioid Receptor Binding Assay
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Cell membranes (400 µg) were incubated with the radioisotope ligand, 2 nM [3H]-diprenorphine (Perkin Elmer), in 25 mM HEPES, pH 7.4, along with or without 1 µM unlabeled kappa agonist, U50, 488H (Sigma, St. Louis, MO, USA), for 10 min at RT. One µM of DADLE [d -Ala2, d -Leu5]-enkephalin and DAMGO (Sigma) were also included in every reaction in order to block the delta and mu opioid receptor bindings. The binding was stopped by the filtration method using GF/B filters. The filters were washed with 7% PEG in HEPES buffer, incubated with Scintiverse-BD (Fisher Scientific, Waltham, MA, USA) for 1 h at RT, and then subjected to counting using a beta-counter (Beckman Coulter, Brea, CA, USA). Data was normalized using the amount of proteins in control and treated cells.
4
Cardiomyocyte Hypertrophy Assay Protocol
5
Measuring Cell Proliferation by [3H]Thymidine Incorporation
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[3H]thymidine incorporation assay was performed in the cells cultured as reported above; the cells were incubated for the last 2 h in the presence of methyl [3H]thymidine (1–2 µCi/ml) (Perkin Elmer, Monza, Italy) and then processed using trichloroacetic acid precipitation as reported previously [41] (link). The recovered radioactivity was measured in a beta counter (Beckman Coulter s.r.l., Milano, Italy).
6
Triglyceride Quantification in Drosophila
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To measure TAG content, we used a triglyceride extraction method as previously described (FOLCH et al. 1957 (link)). Six to ten replicates of six 3–5-day old flies were collected and washed in four dilutions (1:2,1:4,1:10,1:20) of isopropanol to remove excess food. The aqueous phase was used for glucose assays. The lipids were collected after extraction, evaporated under N2 gas and reconstituted with 95% ethanol. Scintillant was added to samples with radioactive tracer instead of ethanol and analyzed on a beta counter (Beckman Coulter). For nonradioactive samples, samples were spun and placed into 96 well plates (Sigma-Aldrich, # CLS9018BC) and incubated with triglyceride reagent (200 µl; Thermo Fischer Cat #TR22421) at 37°C for 30 minutes. Precimat glycerol reagent (Thermo Fischer # NC0091901) was used as a standard. Total absorbance at 500 nm was measured in a plate reader (Beckman) and subtracted from a blank before determining the amount of triglyceride using the reference standard curve. All calculations were performed in Excel (Microsoft) and graphed in Prism.
7
Quantifying De Novo Lipogenesis in HuH-7 Cells
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For de novo lipogenesis, HuH‐7 cells were labeled with 1 µCi/ml of 14C‐Acetate for 1 h. Lipid was extracted as described above and separated on TLC. The mobile phase used for lipid separation consists of hexane:diethyl ether:acetic acid (170:30:1) and visualized using iodine. The plasma lipids (PLs), fatty acid (FA), TGs, and cholesterol ester spots were scraped, and the counts in each spot were measured using a beta counter (Beckman Coulter). Counts were measured and normalized to protein from respective wells as measured by BCA.
8
Splenocyte Cytokine Response to SG Autoantigen
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At day 63, spleens were isolated and stimulated with homogenized SG autoantigen (50 μg/mL) in a flat-bottom 96-well plate (Corning Inc. Corning, NY, USA) for 96 h, and then the cells were pulsed with [3H] thymidine for 18 h before harvesting. The incorporation of radioactivity was measured in a beta-counter (Beckman Instruments). The IFN-γ and IL-17A production in the cell supernatants was determined by ELISA.
For intracellular detection of cytokines, mouse spleen cells were incubated with SG autoantigen for 48 h. GolgiStop (BD Biosciences, San Diego, CA, USA) solution was added 6 h before harvesting the cultured cells. The cells were then washed twice in FACS buffer and stained with phycoerythrin-conjugated anti-mouse CD4 (BioLegend, San Diego, CA, USA). Splenocytes were fixed and subjected to intracellular staining using the Cytofix/Cytoperm Plus Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). FITC-conjugated mAbs specific to murine IFN-γ and IL-17A were purchased from BioLegend (San Diego, CA, USA). All samples were detected on an Accuri C5 cytometer using C6 Accuri system software (Accuri Cytometers Inc., Ann Arbor, MI, USA).
For intracellular detection of cytokines, mouse spleen cells were incubated with SG autoantigen for 48 h. GolgiStop (BD Biosciences, San Diego, CA, USA) solution was added 6 h before harvesting the cultured cells. The cells were then washed twice in FACS buffer and stained with phycoerythrin-conjugated anti-mouse CD4 (BioLegend, San Diego, CA, USA). Splenocytes were fixed and subjected to intracellular staining using the Cytofix/Cytoperm Plus Kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). FITC-conjugated mAbs specific to murine IFN-γ and IL-17A were purchased from BioLegend (San Diego, CA, USA). All samples were detected on an Accuri C5 cytometer using C6 Accuri system software (Accuri Cytometers Inc., Ann Arbor, MI, USA).
9
Quantification of Triglyceride Synthesis
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Total TG was measured by colorimetric enzymatic assay. Briefly, lipids were extracted from day 20 HLCs and organoids using (3:2) hexane:isopropanol, and pellet was resuspended in water after 15% Triton‐chloroform treatment. Absorbance was measured at 500 nm after incubating 10 µL of extracted lipid or 10 µL of media with 190 µL TG Infinity enzymatic solution for 15 minutes at 37°C (Thermo Fisher Scientific). To measure newly synthesized TGs, day 20 cells were pre‐incubated with [3H]‐1,2,3‐Glycerol (10 µCi/mL) in the presence of Oleic Acid (0.4 mM) for 4 hours. To measure radiolabeled TG, lipids were extracted from cells, fractionated by thin‐layer chromatography, and quantified by beta counter (Beckman Coulter, Brea, CA). TG counts in cells was normalized to the total cellular protein for 2D HLCs, as measured by BCA. For organoids, equal number and sized organoids per treatment were used for the assay.(50 )
10
2-DG Uptake Assay for Insulin Resistance
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2-DG uptake assay was performed in HepG2, IR-HepG2, 3T3L1 and L6 cells4 (link). After 24 hr of seeding, cells were treated with or without PST (25 nM) and/or PSTi8 (150 nM) for 20 hr. For IR, HepG2 cells treated with 30 mM glucose [prepared by supplementing D-glucose in HGDMEM (cat no. D5648) medium] for 48 hr15 (link). Cells were serum starved for 4 hr along with PST and/or PSTi8. Then the cells were incubated with insulin (100 nM) for 30 min in 0.5 mL of warm (37 °C) krebs ringer hepes (KRH) buffer. Glucose uptake was initiated by the addition of 0.3 mL of KRH buffer containing 10 µM 2-DG (0.5 µCi/mL 2-[3 H] DG) to each well. It was terminated after 5 min and the cells were solubilized with 0.05 M NaOH, and radioactivity was measured by beta-counter (Beckman Coulter, USA) and normalized by protein content to measure fold change in glucose uptake among treated and untreated (control)19 (link)–21 (link).
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