Cell density was determined using a Neubauer hemacytometer (Hausser Scientific, Horsham, PA, United States). For the comparison between ecotypes, cell densities were determined using a double beam spectrophotometer (Lambda 25, Perkin Elmer). Optical density (OD) was measured at 735 nm and calibration curves prepared for each strain and each condition in order to associate OD to cell density. Calibration curves are reported in Supplementary Figure 1A . Determination of growth rates was obtained using the software CurveExpert Basic1 through a logistic model fitting (Bolzmann).
Neubauer hemacytometer
Manufactured by Hausser Scientific
Sourced in United States
The Neubauer hemacytometer is a laboratory instrument used for the counting and enumeration of cells, such as blood cells or other biological particles, in a sample. It consists of a thick glass slide with a precisely ruled grid pattern that defines a known volume of the sample under observation. The grid allows for accurate counting and calculation of the cell concentration within the sample.
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3 protocols using neubauer hemacytometer
1
Cell Density and Growth Rate Determination
2
Apoptosis Analysis of Treated Cells
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Total viable cells were assessed on a Neubauer hemacytometer (Hausser Scientific, Horsham, PA, USA) using the trypan blue (Life Technologies) exclusion method. Apoptosis analysis was performed using an Apoptosis Detection kit (Thermo Fisher Scientific). After being treated with inhibitors for 24 or 48 h, the cells were pelleted and resuspended in binding buffer with PI and APC-conjugated Annexin V at room temperature for 15 min. Cells were analyzed using a FACS Calibur (BD Bioscience, San Jose, CA, USA). Total apoptotic cell numbers were calculated as the sum of ‘early’ apoptotic cells (Annexin V+ only) and ‘late’ apoptotic cells (Annexin V+/PI+).
3
Identification and Cultivation of B. cinerea
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The phytopathogenic necrotrophic fungus B. cinerea BC2 [50 (link)] was used in this study. The strain identification was confirmed by PCR amplification of a 159-bp segment of the B. cinerea specific marker sequence C729+/C29− [64 (link)] using oligonucleotides BCN1F (5′ CCT GGG TTG TTG CTA TCC TTT ATC 3′) and BCN1R (5′ GGC GTC GTT GGT GAG TGG 3′) [40 (link)]. B. cinerea BC2 was routinely maintained in potato dextrose agar (211,900; BD Bioxon,) plates and incubated at 25 °C in darkness until sporulation. Conidia were collected by adding deionized sterile water to the Petri dishes, and then the suspension was collected with a micropipette. Conidia quantification was performed using a Neubauer hemacytometer (Hausser Scientific, Horsham, PA, USA).
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