Shandon cytocentrifuge

Cytospins were performed with 2 × 10⁴ monocytes/macrophages with a Shandon cytocentrifuge (Thermo Scientific, Langenselbold, Germany). Following May-Grünwald-Giemsa staining, cells were allowed to dry and were covered before evaluation by bright field microscopy. Adjustments in brightness and contrast were performed using the Office Power Point software.

A total of 1 × 10⁴ cells per well were seeded on to a 96 well plate (Sarstedt) and allowed to attach overnight forming a monolayer before being serum starved for 24 h. Cells were then treated with both 40 and 70 μg mL⁻¹ of ASL or vehicle-only control (PBS) for 24 h. Ten micrograms of supernatant was removed from each well, placed into a 1.5 mL microtube (Sarstedt). Cells were stained with 5 μM of both Syto 9 and propidium iodide (Merck) by adding directly into the microtube and incubating at room temperature for 30 min. The cells were then centrifuged at 150 × g for 5 min and the supernatant was aspirated. The remaining cells pellet was washed using ice-cold PBS (pH 7.4) (ThermoFisherScientific) and spun onto a microscope slide using a Shandon cytocentrifuge (ThermoFisherScientific) for 5 min at 176 × g. Slides were subsequently imaged with a Axio Scope 1 microscope (Zeiss) at 400 × magnification. Following staining with Syto9 and propidium iodide, fluorescent staining was present in nucleus therefore live cells appeared green while dead cells fluoresced red (Altman et al. 1993 (link)) and as such cells were counted. A minimum of 1 cell and a maximum of 250 cells were counted per field of view. Experiments were plated in triplicate and repeated three times, data set shown as mean ± standard error of the mean (SEM).

Lymphocytes in CSF were detected by VetScan HM2 (ABAXIS, Union City, CA) according to manufacturer’s instructions. One hundred μl of each CSF sample was used for Giemsa staining as following: cells were attached to glass slides using a Shandon cytocentrifuge (Thermo Fisher Scientific Inc., Waltham, MA) at 1,000 rpm for 10 min and then stained with Giemsa’s Azure Eosin Methylene Blue solution (Merck Millipore, Billerica, MA). Stained cells were analyzed by microscopy.

Light microscopy images were obtained using an ADF 1350B microscope equipped with an ADF Live 4h camera (ADF optics Co., Ltd., Jiaxing, China). Cytospins were prepared using a Shandon cytocentrifuge (Thermo Scientific, Langenselbold, Germany). Briefly, 3–4 × 10³ cells were spun for 3 min at 1000 rpm. Slides were dried, treated with methanol for 5–10 min, stained with Romanovsky–Giemsa stain (Merck, Burlington, MA, USA) for 15–30 min, dried and analyzed using bright-field microscopy.

Light images were obtained using a Zeiss Axioskop 40 microscope equipped with an AxioCamMRc5 camera (Carl Zeiss, Germany). Cytospins were prepared using a Shandon cytocentrifuge (ThermoScientific, Langenselbold, Germany). Briefly, 3–4 × 10³ cells were spun for 3 min at 1,000 rpm. Slides were dried, treated with methanol for 5–10 min, stained with Romanovsky-Giemsa stain (Merck, Germany) for 15–30 min, dried and analyzed using bright-field microscopy.