gondii RH tachyzoites. Two hr post-infection the unrecruited parasites were washed away with PBS. The cells were fixed with polyformaldehyde for 30 min and permeablized with Triton X-100 and blocked with 5% BSA in PBS. The cells were incubated with the primary antibody at 4°C overnight. The coverslips were washed 3 times with PBST, 5 min each wash, and then FITC conjugated secondary antibody was added to the coverslips and incubated for 1 hr at room temperature. The coverslips were washed 3 times with PBST, 5 min each wash, and stained with 10
nM DAPI (Sigma) for 10 min at room temperature, then washed three times with PBS (5 min each wash) with CYT387 slight shaking. The coverslips were rinsed with double distilled water and air-dried. The coverslips were mounted and ready for fluorescence microscopy. Selleck Saracatinib Primary antibodies of monoclonal rabbit
anti-human RhoA antibody (Cell Signaling) and polyclonal rabbit anti- human Rac1 antibody (Abcam) were used in 1:100 dilutions. Secondary antibody of goat anti-Rabbit IgG-FITC (Abcam) was used in 1:500 dilutions. Fluorescence microscopy for overexpressed CFP tagged RhoA and Rac1 COS-7 cells grown on the PRN1371 coverslips were transfected with the CFP-tagged RhoA and Rac1 plasmids for 48 hr, and then infected with tachyzoites for 2 hr. Washed three times with PBS, the cells were fixed in 4% polyformaldehyde for 30 min. After aspiration, cells were stained with 10 nM DAPI (Sigma) for 10 min at room temperature, then washed three times with PBS (5 min each wash) with slight shaking. The coverslips were rinsed with double distilled water, air dried and mounted for fluorescence microscopy. Infection rate counting and statistical analysis 16-HBE cells, mock or transfected with Neg Ctrl siRNA, RhoA siRNA, Rac1 siRNA, and RhoA + Rac1 siRNA were infected with 1 × 105 T. gondii RH tachyzoites per well for 3 hr. The cells were washed 3
times with PBS. After aspiration, cells were stained with Giemsa stain (Sigma) for 10 min, and then washed three times with PBS (5 min each wash) with slight shaking. The coverslips were rinsed with double distilled water and air dried. The COS-7 cells overexpressing the CFP chimeras and the mock cells were also infected with 1 × 105 T. gondii RH tachyzoites Etofibrate per well for 2 hr. The COS-7 mock cells were stained with Giemsa stain as above mentioned. The CFP chimeras overexpressed in COS-7 cells do not need staining. The coverslips were rinsed with double distilled water and air dried. The coverslips were mounted and ready for infection rate counting. For Giemsa stained cells, infection rate was the percentage of T. gondii tachyzoites infected cells among 100 cells randomly selected. In the CFP-tagged overexpressed group, the infection rate was presented as the percentage of those tachyzoites infected fluorescent cells among 100 fluorescent cells randomly selected. The infection rate experiment was performed in triplicate.
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