Keratinocytes had been more incubated for 4 hrs after which contaminated by chan

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Keratinocytes had been more incubated for 4 hrs after which contaminated by chan

Mensagem  jh123 em Seg Maio 02, 2016 10:30 pm

Keratinocytes had been more incubated for 4 hrs after which contaminated by changing the infection medium con taining lentiviral particles, polybrene abt263 and Y 27632 at many concentrations.The infection medium was re moved and replaced by fresh medium after overnight in cubation.The treatment method with Y 27632 continued until Day 4 in culture.Soon after seven days of cultivation, the ex pression level of the transgene was analyzed by Western blotting and flow cytometry.Polybrene and Y 27632 have been obtained from Sigma Aldrich and Wako, respectively.The pro cedure of lentiviral infection and subsequent examination of gene transduction are described in Figure one.Western blotting A complete of 104 keratinocytes had been seeded in a 12 nicely cell culture plate with mitomycin C taken care of three T3 J2 cells, grown for 7 days, and analyzed by Western blotting, as previously described.<br><br>The luminescence signals have been detected through the use of ImageQuant LAS4010.Key antibodies utilised have been as follows, rabbit polyclonal antibody to GFP, and mouse monoclonal antibody to tubulin.Secondary antibodies Adriamycin Topoisomerase 阻害剤 were horseradish peroxidase conjugated goat anti mouse and rabbit IgG.Relative density of EGFP bands was measured with Volocity and nor malized with density of tubulin bands.The values Flow cytometry A complete of 2 × 105 keratinocytes had been seeded inside a 60 mm cell culture dish with mitomycin C treated 3 T3 J2 cells, transduced EGFP cDNA with lentivirus vector in pres ence or absence of Y 27632, and grown for 7 days.<br><br>Trypsinized and resuspended keratinocytes were incu bated with mouse monoclonal antibody against six integ rin for ABT199 one hr on ice.Keratinocytes had been incubated with phycoerythrin conjugated goat polyclonal antibody towards mouse IgG for 1 hr on ice.Right after 3 washes with Hanks Balanced salt option containing calcium and magnesium the cells had been then resuspended in HBSS and ana lyzed by utilizing the FACSCan movement cytometer.were determined depending on final results in 3 independent experiments.Cell proliferation assay A total of 104 keratinocytes were seeded in a 12 well cell culture plate with mitomycin C taken care of 3 T3 J2 cells.Just after seven days of cultivation, keratinocytes had been tryp sinized and stained with 0.4% Trypan blue to determine dead cells.<br><br>Then, the amount of cells was coun ted by using a Countess automated cell counter, which could eradicate the dead cells and big mitomycin C taken care of 3 T3 J2 fibroblasts with irregular shape.The values have been determined determined by success from 3 independent experiments.Immunofluorescence microscopy A total of 2 × 103 keratinocytes were seeded at clonal density in a 35 mm cell culture dish with mitomycin C treated 3 T3 J2 cells, grown for six days, and ana lyzed by immunofluorescence microscopy, as previ ously described.Rat monoclonal antibody to 6 integrin, and mouse monoclonal antibody to p63 and involucrin have been used as major antibodies.Cy3 conjugated goat anti rat IgG and anti mouse IgG have been utilized as secon dary antibodies.Calculation of EGFP favourable keratinocytes and 3 T3 feeder cells The percentages of EGFP positive keratinocytes and three T3 feeder cells had been calculated from the following equations, EGFP constructive keratinocytes × a hundred, and EGFP positive 3 T3 feeder cells × 100.

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