Fc microplate reader

Hemolytic activity of the peptides was assayed against human red blood cells (hRBCs) by measuring the amount of hemoglobin released after treatment^{26 (link)}. The hRBCs, freshly collected from a healthy volunteer in polycarbonate tubes containing heparin, were washed three times in sterile phosphate buffered saline (PBS) and centrifuged at 2,000 × g for 5 min or until the supernatant became clear. The hRBCs were diluted to a final concentration of 2% (vol/vol), then 50 µl of the hRBCs suspension was incubated with 50 µl of different concentrations (0.98 to 250 μg/ml) of a peptide dissolved in PBS. After 1 h of incubation at 37 °C, intact hRBCs were pelleted by centrifugation at 2,000 × g for 10 min. The supernatant was transferred to a new 96-well plate and the release of hemoglobin was monitored by measurement of absorbance at 405 nm using a Multiskan FC microplate reader. The hRBCs in PBS only (OD_Blank) and in 0.1% Triton X-100 (OD_{Triton X-100}) were employed as negative (0% hemolysis) and positive (100% hemolysis) controls, respectively. The percentage of hemolysis was calculated according to the following equation: $$\%\mathrm{Hemolysis}=({\mathrm{OD}}_{\mathrm{Sample}}-{\mathrm{A}}_{\mathrm{Blank}})/({\mathrm{OD}}_{\mathrm{TritonX}-100}-{\mathrm{OD}}_{\mathrm{Blank}})\times 100$$

The hemolytic activity of the 6e, 6f, 6g, 6i, 8d, 8e was assayed against human red blood cells (hRBCs) by measuring the amount of hemoglobin released after treatment. The hRBCs, freshly collected from a healthy volunteer in polycarbonate tubes containing heparin, were washed three times in sterile phosphate-buffered saline (PBS) and centrifuged at 2000×g for 5 min or until the supernatant became clear. The hRBCs were diluted to a final concentration of 2% (vol/vol), then 50 μl of the hRBCs suspension was incubated with 50 μl of different concentrations (0.75 to 100 μg mL⁻¹) of the tested compounds dissolved in PBS. After 1 h of incubation at 37 °C, intact hRBCs were pelleted by centrifugation at 2000×g for 10 min. The supernatant was transferred to a new 96-well plate and the release of hemoglobin was monitored by measurement of absorbance at 405 nm using a Multiskan FC microplate reader. The hRBCs in PBS only (ODBlank) and in 0.1% Triton X-100 (ODTriton X-100) were employed as negative (0% hemolysis) and positive (100% hemolysis) controls, respectively. The percentage of hemolysis was calculated according to the following equation:

The hemolytic activity of peptides was determined as the amount of hemoglobin released from lysed human red blood cells (hRBCs) after treatment with peptide^{20 (link)}. Fresh hRBCs were collected from a healthy volunteer in polycarbonate tubes containing heparin. Then, the collected packed RBCs were washed at least three times (or until the supernatant was clear) with sterile phosphate-buffered saline pH 7.4 (PBS) and centrifuged at 2,000 × g for 5 min. The 2% (v/v) of washed hRBCs in PBS were incubated with serially diluted peptides (0.98 to 250 μg/ml) for 1 h at 37 °C. After centrifugation, the supernatants were monitored for optical density (OD) at 405 nm using a Multiskan FC Microplate Reader^{21 (link)}. Values for 0% (negative control) and 100% (positive control) lysis were determined by incubating the hRBCs with PBS only (OD_Blank) and 0.1% (v/v) Triton X-100 (OD_{Triton X-100}), respectively. The experiments were performed in triplicate. The percent hemolysis was calculated according to the following equation: $$\%\text{Hemolysis}=\left({\text{OD}}_{\text{Sample}}-{\text{OD}}_{\text{Blank}}\right)/\left({\text{OD}}_{\text{Triton X}-100}-{\text{OD}}_{\text{Blank}}\right)\times 100$$
Experiments associated with human volunteers were carried out in accord with the ethical standards of and approved by the Ethics Committee of Thammasat University (COA No. 066/2562). The informed consent was voluntarily written by all individual participants.