Guava easycyte 6 2l

The apoptosis states of HepG2 cells were determined by an Annexin V-FITC/PI apoptosis kit. Cells were collected and washed with ice-cold PBS twice. Then, the cells were resuspended and diluted to 1 × 10⁶ cell/mL with binding buffer. The suspended cells were dyed by 10 μL of Annexin V-FITC for 30 min at room temperature and then stained with five μL of propidium iodide (PI) for 5 min. After incubation, the apoptosis of cells was determined by flow cytometry with Guava® easyCyte 6-2L (Millipore, Billerica, MA, USA).

DiCre recombinase mediated excision of targeted DNA sequences in vivo was achieved by a single oral administration of 200μg rapamycin (1mg/ml stock, Rapamune, Pfizer) to mice. Excision of the GFP cassette in blood stage parasites was monitored by flow cytometry using a Guava EasyCyte 6/2L bench cytometer equipped with 488 nm and 532 nm lasers (Millipore) to detect GFP and mCherry, respectively. To analyze parasite development in the mosquito, rapamycin was administered to infected mice 24 hours prior to transmission to mosquitoes, as described [19 (link)]. Midguts were dissected out at day 14 post infection. The haemolymph was collected by flushing the haemocoel with complete DMEM, day 14 to 16 post infection. Salivary gland sporozoites were collected between 21–28 days post feeding from infected mosquitoes, by hand dissection and homogenization of isolated salivary glands in complete DMEM. Live samples (infected mosquito midguts or salivary glands, sporozoites) were mounted in PBS and visualized live using a Zeiss Axio Observer.Z1 fluorescence microscope equipped with a LD Plan-Neofluar 40x/0.6 Corr Ph2 M27 objective. The exposure time was set according to the positive control and maintained for both untreated and rapamycin-treated parasites, in order to allow comparisons. All images were processed with ImageJ for adjustment of contrast.

A human annexin VFITC(AV)/PI apoptosis kit was used, as in our previously reported method. Briefly, HepG2 cells were inoculated in 12-well plates at a density of 2.0 × 10⁵ cells per well. After the cell incubation with CF, the cells were washed three times with pre-cooled PBS and then collected in 1.5 mL Eppendorf tubes after centrifugation at 1500× g. Then, 100 μL of buffer and 5 μL of Annexin V-FITC and PI were added to the cells and reacted for 10 min in a dark environment. Finally, apoptosis of cells was detected by flow cytometry (Guava easy Cyte 6-2 L, Millipore, Billerica, MA, USA).

Host cell invasion by GFP-expressing sporozoites was monitored by flow cytometry.⁷⁴ (link) Briefly, hepatoma cells (3 × 10⁴ per well in collagen-coated 96-well plates) were incubated with sporozoites (5 × 10³ to 1 × 10⁴ per well). For measurement of cell traversal activity, sporozoites were incubated with cells in the presence of 0.5 mg/ml rhodamine-conjugated dextran (Life Technologies). At different time points ranging from 15 minutes to 3 hours post-infection, cell cultures were washed, trypsinized and analyzed on a Guava EasyCyte 6/2L bench cytometer equipped with 488 nm and 532 nm lasers (Millipore), for detection of GFP-positive cells and dextran-positive cells, respectively, to measure total invasion rates and cell traversal activity. To assess liver stage development, HepG2 or HepG2/CD81 cells were infected with GFP-expressing sporozoites and cultured for 24-36 hours before analysis either by FACS or by fluorescence microscopy, after fixation with 4% Formaldehyde (FA) and labeling with antibodies specific for UIS4 (Sicgen).

H₂O₂-induced HepG2 cells were employed to determine the inhibitory effect on ROS production [3 (link)]. HepG2 cells (1.0 × 10⁵ cells per well) were seeded in a 6-well plate and co-cultured with isolated compounds with 50 μg/mL and Vc (8 μg/mL). After incubation for 20 h, the medium was removed, and 2 μL of H₂O₂ (0.5 mM) was added to each well for another 6 h. At the end of experiment, the cells were labeled with 2 μL DCFH-DA (10 mM) in the dark at 37 °C for 20 min. The absorbance was recorded by flow cytometry (Guava easyCyte 6-2L, Millipore, Billerica, Massachusetts, MA, USA).

The annexin V-FITC/PI apoptosis kit (Beijing Sizhengbai Biotech Co., Ltd., Beijing, China) was used to detect the HepG2 cell apoptosis [10 (link)]. HepG2 cells were pre-cultivated for 20 h with or without the test compounds, and then incubated with APAP at a concentration of 10 mM for another 20 h. The cells were collected, washed with pre-cooled PBS, and resuspended in 100 μL of binding buffer. The cells were then cultured with the annexin V-FITC (10 μL) for 10 min in the dark and stained with propidium iodide (PI) (5 μL) for 5 min in an ice bath. By using the flow cytometry (Guava^® easyCyte 6-2L, Millipore, Billerica, MA, USA), cell apoptosis was detected.