Culture were pelleted by centrifugation at 1000 rpm for 10 min and incubated with a protoplastization solution consisting of 10 mM MES buffer pH 5.8, 10 mM CaCl2, 0.4 M mannitol, 1 Macerozyme and 1 Cellulase (for about 1 g of cells 5 mL enzymatic solution was added) at room temperature in the dark for 3? hours under gentle agitation. After incubation the protoplasts were sieved through a 90 m mesh without applying pressure. 200 L of protoplasts were mixed with 200 L of 0.75 LMP agarose (at 3 ) and 80 L aliquots were placed on a microscope slide previously coated with 0.75 agarose. A 22?2 mm glass cover slip was placed on each gel and the slides were allowed to set on ice for a few minutes, the coverslips were then removed. The slides were marked as “control” (protoplasts from GW0742 chemical information cultures with no treatment), “heat treated” (protoplasts treated for 20 min at 50 ), “10 nM, 50 nM or 100 nM” (protoplast from cultures treated with one of the three QD concentrations), “buffer” (protoplasts from cultures treated with one of the three QD concentrations plus enzyme buffer), “FPG” (protoplasts from cultures treated with one of the three QD concentrations plus FPG enzyme) and “Endo III” (protoplasts from cultures treated with one of the three QD concentrations plus Endo III enzyme).Alkaline unwinding/neutral electrophoresisplaced in 0.3 M NaOH and 1 mM EDTA, pH approximately 13,0 at 4 for 20 minutes. The samples were then neutralized by dipping PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 in a 0.4 M Tris Cl, pH 7.5 solution, 3 times for 5 minutes at 4 . The slides were transferred to the electrophoresis tank and placed in TBE (pH 8) for a few minutes and then electrophoresed for 10 min at 25 V, 10 mA at 4 . After being electrophoresed they were fixed in ethanol 70 2×5 min and left to dry overnight. 20 L of 1 g/ mL DAPI was placed on each gel and covered with a coverslip, and scored after 5 min.Neutral incubation/ neutral electrophoresisDNA order trans-4-Hydroxytamoxifen unwinding and electrophoresis at neutral pH (pH 7?) facilitates the detection of double-strand breaks and crosslinks. Under these conditions the total DNA damage is much less pronounced than under alkaline conditions [45]. In brief, slides marked as “control”, “heat treated” and “10 nM, 50 nM or 100 nM” were lysed in the Coplin jar for 1 hour at 4 in 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris?HCl pH 7.5. They were then equilibrated in TBE 2 times for 5 min and electrophoresed in TBE 10 min at 25 V, 10 mA. They were fixed, stained as above and scored.Scoring for DNA damageThe modification of the comet assay described by Angelis et al. [44] employs various combinations of neutral and alkaline solutions immediately prior to and during electrophoresis. Exposure of DNA to highly basic conditions prior to electrophoresis under neutral conditions (N/A protocol) allows for the preferential detection of DNA SSBs. Briefly, cells embedded in agarose were lysed in a Coplin jar for 1 hour in 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris Cl pH 10, 1 Triton X-100 at 4 . The slides marked with “buffer”, “FPG” and “EndoIII” were then washed 3 times for 5 minutes at 4 with enzyme buffer containing 40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL BSA, pH 8 adjusted with KOH. After the last wash the excess of liquid was drained with the tissue and the slides were placed on ice. Then 50 L of enzyme buffer, FPG (104 dilution) or Endo III (104 dilution) were added to the respective gels and covered with a coverslip. The slides were then transferred to a moistening box and incubated at 37 for.Culture were pelleted by centrifugation at 1000 rpm for 10 min and incubated with a protoplastization solution consisting of 10 mM MES buffer pH 5.8, 10 mM CaCl2, 0.4 M mannitol, 1 Macerozyme and 1 Cellulase (for about 1 g of cells 5 mL enzymatic solution was added) at room temperature in the dark for 3? hours under gentle agitation. After incubation the protoplasts were sieved through a 90 m mesh without applying pressure. 200 L of protoplasts were mixed with 200 L of 0.75 LMP agarose (at 3 ) and 80 L aliquots were placed on a microscope slide previously coated with 0.75 agarose. A 22?2 mm glass cover slip was placed on each gel and the slides were allowed to set on ice for a few minutes, the coverslips were then removed. The slides were marked as “control” (protoplasts from cultures with no treatment), “heat treated” (protoplasts treated for 20 min at 50 ), “10 nM, 50 nM or 100 nM” (protoplast from cultures treated with one of the three QD concentrations), “buffer” (protoplasts from cultures treated with one of the three QD concentrations plus enzyme buffer), “FPG” (protoplasts from cultures treated with one of the three QD concentrations plus FPG enzyme) and “Endo III” (protoplasts from cultures treated with one of the three QD concentrations plus Endo III enzyme).Alkaline unwinding/neutral electrophoresisplaced in 0.3 M NaOH and 1 mM EDTA, pH approximately 13,0 at 4 for 20 minutes. The samples were then neutralized by dipping PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 in a 0.4 M Tris Cl, pH 7.5 solution, 3 times for 5 minutes at 4 . The slides were transferred to the electrophoresis tank and placed in TBE (pH 8) for a few minutes and then electrophoresed for 10 min at 25 V, 10 mA at 4 . After being electrophoresed they were fixed in ethanol 70 2×5 min and left to dry overnight. 20 L of 1 g/ mL DAPI was placed on each gel and covered with a coverslip, and scored after 5 min.Neutral incubation/ neutral electrophoresisDNA unwinding and electrophoresis at neutral pH (pH 7?) facilitates the detection of double-strand breaks and crosslinks. Under these conditions the total DNA damage is much less pronounced than under alkaline conditions [45]. In brief, slides marked as “control”, “heat treated” and “10 nM, 50 nM or 100 nM” were lysed in the Coplin jar for 1 hour at 4 in 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris?HCl pH 7.5. They were then equilibrated in TBE 2 times for 5 min and electrophoresed in TBE 10 min at 25 V, 10 mA. They were fixed, stained as above and scored.Scoring for DNA damageThe modification of the comet assay described by Angelis et al. [44] employs various combinations of neutral and alkaline solutions immediately prior to and during electrophoresis. Exposure of DNA to highly basic conditions prior to electrophoresis under neutral conditions (N/A protocol) allows for the preferential detection of DNA SSBs. Briefly, cells embedded in agarose were lysed in a Coplin jar for 1 hour in 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris Cl pH 10, 1 Triton X-100 at 4 . The slides marked with “buffer”, “FPG” and “EndoIII” were then washed 3 times for 5 minutes at 4 with enzyme buffer containing 40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL BSA, pH 8 adjusted with KOH. After the last wash the excess of liquid was drained with the tissue and the slides were placed on ice. Then 50 L of enzyme buffer, FPG (104 dilution) or Endo III (104 dilution) were added to the respective gels and covered with a coverslip. The slides were then transferred to a moistening box and incubated at 37 for.
