All in one fluorescence microscope bz x700 series

Time course microscopic observations of virus infection were done on a Keyence All-in-one Fluorescence Microscope BZ-X700 Series. ADSCs were engineered to express eGFP and were followed on the GFP channel (1 s exposure) while virus infection with the TurboFP635-engineered L14 virus was monitored on the TRITC channel (3 s exposure). Images at 4× or 10× magnification were collected and overlaid with bright field (phase contrast, 1/50 s exposure).

The cellular localization of BmP53 was determined by an Immunofluorescence antibody assay (IFA) according to [14 (link)]. Briefly, thin blood smears of B. microti-infected erythrocytes or non-infected isolated hamster platelets were fixed in a solution of 95% methanol and 5% acetone (v:v) at -20°C for 30 min and then probed with mouse anti-rGST-BmP53tr1-TSP1 immune sera diluted in 4% fetal bovine serum in PBS (1:100) for 1 h at 37°C in a moist chamber. A secondary antibody of goat anti-mouse or -Rabbit IgG (H+L) (Alexa-Fluor 488 conjugate) (green dye) and goat anti-mouse IgG (H+L) (Alexa-Fluor 594-conjugate) (red dye) (Life Technologies, USA) diluted in the same buffer (1:250) was applied on the smears and then incubated for 1 h at 37°C. DNA was stained with Hoechst blue dye (Dojindo Molecular Technologies, Inc., USA) in PBS (1:250) for 10 min at RT. After washing with PBS-T, the smears were mounted using a fluorescent mounting medium (Dako, USA) and then covered with a glass coverslip. Imaging was performed using All-in-One Fluorescence Microscope, BZ-X700 series (KEYENCE corporation, Japan). For the co-localization study, anti-rBmSA1 rabbit serum [43 (link)] was used as a specific marker for B. microti Gray strain parasites.

RM20 adipose-derived stem cells at passage 0 were engineered to express eGFP under the control of the CMV promoter. A Lentiviral vector (VectorBuilder) containing eGFP was used to introduce eGFP for constitutive expression. 10,000 eGFP-positive cells were sorted at passage 1 and subsequently at passage 2 using the BioRAD S3 Cell Sorter. eGFP expression was confirmed by flow cytometry and fluorescence microscopy using the Keyence All-in-one Fluorescence Microscope BZ-X700 Series.