Easycyte mini system

Strains were exposed to OGME (MIC, 2MIC and 4MIC), to the RPMI culture medium (negative control) and to amphotericin B (positive control, 4 μg/mL) for cell viability determination. Treated cells were incubated at 35 °C for 24 h. Subsequently, aliquots were collected at 3, 6, 12 and 24 h time intervals after exposure to the compounds. All experiments were performed in triplicate on 3 independent occasions. Cells were labeled with PI and analyzed by flow cytometry (Guava EasyCyte™ Mini System). For each experiment, 10,000 events were analyzed [29 (link)].

Pulmonary leukocytes were obtained as previously described by Oliveira-Brito et al. [69 (link)]. Prior to phenotyping the cell populations in the lungs, the concentrations of the cell suspensions were determined using a Neubauer chamber. Each sample was adjusted to a concentration of 1 × 10⁶ cells/mL, and the cells were thereafter stained with fluorophore-conjugated antibodies. The following antibodies were obtained from BD Pharmingen (San Diego, CA, USA): anti-CD3 (PE-Cy5 rat anti-mouse CD3, clone 17A2), anti-CD11c (Alexa Fluor 488 rat anti-mouse CD11c, clone M1/70), anti-Ly6G (PE rat anti-mouse Ly-6G, clone 1A8), and anti-CD11b (PE rat anti-mouse CD11b, clone M1/70). After incubation for 30 min at 4 °C with 0.5 μg of anti-CD16/CD32 mAb (Fc block, clone 2.4G2, BD Pharmingen), 2.5 μg/mL of the aforementioned antibodies was added and the suspension incubated for 45 min at 4 °C. The cells were washed twice with PBS and fixed with 1% PBS-formaldehyde for further analysis using flow cytometry (Guava EasyCyte™ Mini System).

Raji and Daudi cells (1 × 10⁶ cells/mL) were incubated, separately, with the following pharmacological inhibitors: SB202190 (p38MAPK inhibitor), SP600125 (JNK inhibitor), PD98059 (p42/44MAPK inhibitor), H-7 (PKC inhibitor), Genistein (PTK inhibitor), Z-VAD (caspases inhibitor), Torin-1 (mTOR inhibitor), R406 (Syk inhibitor), NQ-301 (inhibitor of CD45 phosphatase activity on Lck) and Dasatinib (inhibitor of Lyn, Fyn, and Blk) for 210 min at 37 °C. The cells were subsequently stimulated with ArtinM at concentrations of 1.25 µg/mL (Raji cells), and 20.0 µg/mL (Daudi cells), for 24 or 48 h at 37 °C. Arsenic trioxide (AS₂O₃), at concentrations of 12 µM (Raji cells) and 24 µM (Daudi cells), was used as a positive control, and the medium alone was used as a negative control. The frequency of positive cells for AnV binding and/or PI incorporation was quantified by flow cytometry (Guava EasyCyte™ Mini System), as described in Section 4.9.