Giemsa

May‐Grunwald‐Giemsa staining was performed following the published protocols.⁴¹ Briefly, cells were first smeared onto glass slides, air‐dried, fixed in 100% Methanol for 3‐10 minutes and then went through 2‐step staining: first with 50% (v/v) May‐Grunwald (Sigma, CAS NO. 63590) in phosphate‐buffered saline (pH 6.8) for 3‐5 minutes, then with 10% Giemsa (Sigma, CAS NO. 48900) for 10‐30 minutes and washed with running water for 1‐3 minutes. Images of air‐dried slides were taken under a Leica microscope.

Thick and thin blood smears were prepared from each specimen, in accordance with WHO recommendations, and were analyzed by means of light microscopy [30 ]. The blood smears were fixed with methanol and stained with Giemsa (Sigma-Aldrich, St. Louis, USA). The thick blood smears were pre-stained with buffered methylene blue solution and stained with Giemsa (Sigma-Aldrich, St. Louis, USA) [27 ].

Inhibition of virus replication. Vero cells seeded in 12-well or 24-well plates one day before the experiment were mock treated or treated with TMPyP3-C₁₇H₃₅ at different concentrations (9.6 nM to 4.8 µM) and incubated for 30 min, after which the compound containing medium was replaced with fresh medium and the cells were mock infected or infected with HSV-1 at the indicated multiplicity of infection (MOI). After 30 min, the infected cells were exposed to light or kept in the dark for 15 min. One hour after infection, the infectious media were replaced with fresh growth medium, and the cells were further incubated at 37 °C and 5% CO₂. At the indicated time points, the cells were fixed in 5% MetOH and 10% HAc solution and stained with Giemsa (5% Giemsa, Sigma, Singapore), and the number of plaque was determined, or supernatants were collected, and the viral titer was determined using a standard plaque assay [30 (link)].
Direct effect of TMPyP3-C₁₇H₃₅ on virus particle. A dilution of a virus stock in growth medium (~1 × 10⁷ Pfu) was mock treated or treated with TMPyP3-C₁₇H₃₅ at final concentrations of 4.8 μM, 1.2 μM, 480 nm, 240 nM, 48 nM, or 9.6 nM and exposed to light for 15 min or kept in the dark. The infectious virus in treated samples was assayed by a standard plaque assay [30 (link)].

The Vero cell line was cultivated on the glass coverslips of 10 dishes (35 mm cell culture dish) until confluent. The cells on five coverslips were infected with 1 × 10⁴ tachyzoites of T. gondii and incubated for 4 h, then the glass coverslips were washed with phosphate- buffered saline solution (Gibco BRL). Following 24 h incubation, the three prepared nano-formulations, sulfadiazine (positive control), and RPMI media (negative control) were added to five dishes in a dose of 50 µg/mL for 24 h at 37 °C. Finally, the glass coverslips were washed twice and fixed with methanol prior to staining with Giemsa (Sigma-Aldrich Corp, Saint Louis, MO, USA). Another five dishes were incubated with the three nanoformulae, sulfadiazine, and RPMI media for 24 h. After incubation, the glass coverslips were washed twice, fixed with methanol, and stained with Giemsa (Sigma-Aldrich Corp). All the prepared samples were observed under a light microscope. Qualitative analysis methods were used to observe the effect of the tested nanoformulae on both non-infected and infected cell cultures in comparison to positive and negative controls [39 (link)].