After indicated uptake times, cells were washed three times with binding buffer and lysed in 1 M NaOH.
After indicated times, the cells were washed three times with 0.1 M PBS, then harvested by trypsinization, centrifuged, and resuspended in 0.1 M PBS or 0.5 ml of 1% agarose in Eppendorf tubes.
After indicated treatments, PerMΦs were harvested as described above.
After indicated stimulation, the cells were fixed with 4% paraformaldehyde in PBS and incubated with 0.1 M ammonium chloride (10 min).
After indicated incubation at 37°C, ear sheet halves were removed, the epidermal sheet was separated from the dermis using ammonium thiocyanate and staining was performed.
After indicated treatment times, TUNEL assays for DNA fragmentation were performed using the TUNEL/ApoBRDU assay kit (Invitrogen) according to the manufacturer's protocol.
After indicated time points, medium with non-adhered cells was discarded and wells were gently washed twice with PBS to remove any loosely attached cells.
After indicated treatment times, the Caspase 3/7-Glo Assay (Promega) was performed according to the manufacturer's protocol and bioluminescence measurements were obtained from and Infinite 200 plate reader (Tecan).
After indicated treatments, cells were washed twice in PBS and cell lysates were prepared in PBS containing 1% TX-100, supplemented with 1 mM PMSF and a protease inhibitor cocktail.
After indicated time points, blood, liver, kidney, heart, spleen, brain and other organs were collected as per the approval of the IAEC and drug was extracted by silver nitrate and methanol method.
