Thermo shandon cytospin 4 centrifuge

For microscopic analysis of cytospins, 5 × 10⁴ cells were centrifuged for 10 min at 800 rpm on microscopic slides using the Thermo Shandon Cytospin 4 centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). For the Pappenheim staining, cells were stained 5 min in May-Grünwald dye, washed, and then stained for 20 min in Giemsa dye (Sigma-Aldrich). Cytospins were visualized with a NanoZoomer S210 digital slide scanner (Hamamatsu, Hamamatsu City, Japan) using 40× magnification. Cell morphology was assessed with the NDP.view2 2.8.24 viewing software U12388-01 (Hamamatsu).

2 × 10⁴ − 2 × 10⁵ cells were centrifuged in 150 µl PBS for 10 min at 800 rpm on microscope slides using a Thermo Shandon Cytospin 4 centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Cytospins were stained after Pappenheim (Giemsa/May-Grünwald): 5 min in May-Grünwald followed by 20 min in Giemsa staining solutions (Sigma-Aldrich, Steinheim, Germany). The cytospins were digitised at a 40 × magnification using the Hamamatsu NDP.scan software (version 3.2.17) with the Hamamatsu NanoZoomer S210 digital slide scanner equipped with a Nikon plan apochromat 20x (NA 0.75) objective lens and a colour CMOS sensor type 1/2.3 camera (pixel 4000 × 3000). Morphology was assessed using NDP.view 2.8.24 viewing software U12388-01 https://www.hamamatsu.com/eu/en/product/type/U12388-01/index.html by examination of digitised cytospins; a 90–200-cell differential count was performed. Any cell which did not fit a definition exactly was counted with the category, which it most closely resembled^{32 (link)}. Representative pictures of digitised cytospins or light microscope pictures of cytospins (BX51 microscope, camera XC50, software Cell F version 3.4, all Olympus) are presented.