May gr nwald giemsa stain

Sputum induction and processing were performed at 6 h from the start of exposure in control and high ACR exposure as previously described (Strandberg et al., 2008 (link)). Briefly, the sputum weight was determined and an equal volume of dithiothreitol 0.1% (Sputolysin® reagent, Calbiochem, San Diego, CA) was added to the whole sputum sample and rocked for 15–20 min in a 37 °C in a shaking water bath. The sample was centrifuged (10 min at 280 g). The cell pellet was re-suspended in 2 ml phosphate-buffered saline and put on ice. Total cell count and viability tests with Türk (Histolab Products AB, Gothenburg, Sweden) and Trypan blue (Sigma Aldrich, Steinheim, Germany) were performed. Cytocentrifuge-prepared slides were stained with May-Grünwald Giemsa stain (Sigma Aldrich, Steinheim, Germany) and 300 cells (squamous cells excluded) were assessed for differential cell counts. The sputum samples were included in the analysis if they contained less than 30% squamous cells. The supernatant was dispensed into aliquots and kept in −70 °C until analysis. IL-6 and interleukin-8 (IL-8) were analyzed in supernatant of the sputum. They were analyzed by using Human IL-6 Quantikine® HS Elisa High Sensitivity kit and Human CXCL/IL-8 duo set from R&D Systems® (Minneapolis, MN). The LOD were 0.016 pg/ml and 31.2 pg/ml for IL-6 and IL-8, respectively.

4T1 cells with a density of 2 × 10⁵ cells/well were grown into the six-well plate and incubated until confluent 80%. The cells were then treated with selected concentrations of piperine and PGV-1. After being incubated for 24 h, the cells were rinsed with PBS two times, and the May-Grünwald-Giemsa stain (0.25% w/v, in methanol) (Sigma-USA) was added to each well up to 5 min. After that, the cells were flushed with phosphate buffer pH 7.2 for 1.5 min, and a Giemsa stain diluted with phosphate buffer (1: 50) was added to the well. After waiting for about 15 to 20 min at room temperature, the cells were rinsed briefly by deionized water and finally air-dried. Observations were made using an inverted microscope (Leica) Mitotic catastrophe analysis is indicated by cells with micronucleus or polynuclear (3 (link), 11 (link)). The percentage of mitotic catastrophe cells was calculated by two people with blinded observation method.