Reaction mixtures (5 μl total volume) were prepared according to (Scietti et al., 2018 (link)) by sequentially adding LH3/PLOD3 at 0.2 mg/ml, 0–1 mM peptide substrate or 4 mg/ml gelatin in water (solubilized through heating denaturation at 95°C for 10 min), 500 μM ascorbate, 100 μM 2-OG, variable concentrations of FeCl2 (0–200 μM) and let incubate for 1 h at 37°C. Reactions were stopped by heating samples at 95°C for 2 min prior to transfer into Proxiplate white 384-well plates (Perkin-Elmer), then 5 μl of the Succinate-Glo reagent I (Promega) were added and let incubate 1 h at 25°C, after that 10 μl of the Succinate-Glo reagent II (Promega) were added and let incubate 10 min at 25°C. The plates were then transferred into a GloMax Discovery plate reader (Promega) configured according to manufacturer’s instructions for luminescence detection. All experiments were performed in triplicates. Control experiments were performed using identical conditions by selectively removing LH3/PLOD3, 2-OG or peptide substrates. Data were analyzed and plotted using Prism 7 (Graphpad Software). Statistical evaluations based on pair sample comparisons between uncoupled and coupled assay values using Student’s t-test in Prism 7 (Graphpad software).
Glomax discovery plate reader
Manufactured by Promega
The GloMax Discovery plate reader is a versatile and efficient instrument designed for a wide range of luminescence-based assays. It features a high-sensitivity detection system and a compact, user-friendly design, enabling researchers to perform reliable and accurate measurements in a variety of experimental settings.
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Lab products found in correlation
Hoechst 3342, by Thermo Fisher Scientific (2 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (2 mentions)
Anti gfpandanti h3k36me3, by Abcam (2 mentions)
780 confocal microscope system, by Zeiss (2 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (2 mentions)
Proxiplate white 384 well plates, by PerkinElmer (1 mentions)
Succinate glo reagent 1, by Promega (1 mentions)
Succinate glo reagent 2, by Promega (1 mentions)
Prism 7, by GraphPad (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
Injector system, by Promega (1 mentions)
5 protocols using glomax discovery plate reader
1
Colorimetric Assay for Lysine Hydroxylase Activity
2
SETD2 Methyltransferase Regulation of Cell Proliferation
3
SETD2 Methyltransferase Regulation of Cell Proliferation
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For microscopy, SF11435 cells were grown on glass coverslips and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 70% confluency. Forty-eight hours post transfection, cells were fixed in 4% PFA for 8 minutes, blocked in 2.5% FBS, 200mM glycine, and 0.1% Triton X-100 in PBS for 30 minutes, incubated with anti-GFPandanti-H3K36me3 (Abcam, Cambridge, UK) primary antibodies overnight at 4°C, washed, and incubated with Alexa Fluor secondary antibodies (Thermo Fischer Scientific, Waltham, MA) for 1 hour at room temperature. Hoechst 3342 (Life Technologies) was added to the secondary antibody inclubation to mark DNA. Following a final wash, cells were mounted in ProLong Diamond Antifade Mountant (Thermo Fischer Scientific), and immunofluorescence images were collected using a Zeiss 780 confocal microscope system (Carl Zeiss AG, Oberkochen, Germany).
For cell proliferation assays, SF11435 cells were grown in a 96-well plate and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 50% confluency. Forty-eight hours post-transfection, cell proliferation was assayed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and a GloMax Discovery plate reader per the manufacturer’s protocols (Promega, Madison, WI).
For cell proliferation assays, SF11435 cells were grown in a 96-well plate and transduced with either wild-type SETD2-EGFP or SETD2K2R-EGFP using polybrene at 50% confluency. Forty-eight hours post-transfection, cell proliferation was assayed using the CellTiter 96 Non-Radioactive Cell Proliferation Assay and a GloMax Discovery plate reader per the manufacturer’s protocols (Promega, Madison, WI).
4
Calcium Flux Assay for HL60 Cells
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Agonist-stimulated increase in intracellular calcium concentration was measured using the Fluo-4 NW Calcium Assay Kit by Molecular Probes (Invitrogen) according to the manufacturer’s protocol with modifications. Differentiated HL60 cells were untreated, treated with etomoxir (10 μM), etomoxir with octanoic acid (100 μM) or octanoic acid (100 μM) alone for 2 h at 37 °C. Cells were pelleted, washed once with PBS, and resuspended in calcium assay buffer at a concentration of 2.5 × 106 cells per mL. In total, 125,000 cells (50 μL) per well were plated in a black-walled clear bottom 96-well plate and incubated at 37 °C and 5% CO2 for 30 min to settle the cells. In total, 50 μL of the 2× dye loading solution was added to each plate and incubated at 37 °C for 30 min, then at room temperature for an additional 30 min. Fluorescence (excitation 494 and emission 516 nm) was measured for 40 s continuously after adding fMLF (10 nM) to each well using the Glo Max Discovery plate reader with an Injector System (Promega Corporation, Madison, WI).
5
Transwell Assay for T Cell Migration
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Peripheral blood lymphocytes were isolated from the human blood of healthy volunteers76 . A Polymorph density gradient (Accurate Chemical & Scientific Corporation, #AN221725) was used to isolate peripheral blood mononuclear cells that were subsequently selected and differentiated into T cell lymphocytes in Roswell Park Memorial Institute 1640 media (Life Technologies, #11875093) supplemented with 10% FBS, 1% penicillin/streptomycin, phytohemagglutinin 1 μg/ml (10576015, Thermo Fisher Scientific) and recombinant human IL-2 20 ng/ml (202-IL, R&D Systems). The QCM Leukocyte Migration Assay (MilliporeSigma, # ECM557) and a GloMax Discovery plate reader (Promega, #PAGM3000) were used to quantify transwell T cell lymphocyte migration from the apical chamber over 4 h at 37 °C. Unconditioned HEI-193 media served as a negative control, unconditioned HEI-193 media supplemented with recombinant human CCL21 600 ng/ml (366-6C, R&D Systems) served as a positive control, HEI-193 media 5 days after 12.5 Gy in 1 fraction, or HEI-193 media without radiation were placed in the basolateral chamber. All conditions were free from serum or other supplements unless specifically indicated.
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