Xm10 camera

Epimastigote samples from the days 1-7 after transfection were washed twice with PBS. After letting the cells settle for 30 min at room temperature onto poly-L-lysine coated coverslips, parasites were fixed at room temperature for 20 min with 4% formaldehyde in PBS, followed by a cold methanol treatment for 5 min. Parasites were washed with PBS and incubated with polyclonal TcNDPK3 antibodies (1 : 200), followed by Alexa Fluor ® 488-conjugated secondary antibody and slides were mounted using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were observed in an Olympus BX60 fluorescence microscope. Images were recorded with an Olympus XM10 camera.

Intracellular Ca 2+ mobilization was measured as described by our group earlier. Confluent layers of HUVECs were cultured in 96-well plates for 24h followed by pretreatment with 400 μM of CoCl 2 for 4 h or 24 h. The cells were then loaded with 2 μM of Fluo-4-AM for 20 min, followed by a 20 min incubation in HBSS. Fluorescence microscopy was performed using an Olympus XM-10 camera. Baseline fluorescence was determined by taking three photos before adding 0.6 μM of rMASP-1, followed by sequential images every 5 s for 2 min. Changes in fluorescence intensity were calculated by analyzing at least 20 cells per image using CellP software. We used 50 μM of Histamine as a positive control.

For CREB experiments, HUVECs were treated with 0.6 μM of rMASP-1, 400 μM of CoCl 2 , or both for 2 h with or without 6 μM of C1INH. A second plate of HUVECs was placed in a hypoxic (1% O 2 ) incubator with or without 0.6 μM of rMASP-1 treatment for 2 h. In another experimental setup, HUVECs/HUAECs were treated with varying concentrations of rMASP-1 (0.06 μM, 0.2 μM, 0.6 μM, 2 μM), 400 μM of CoCl 2 , or the combination of each dose of rMASP-1 with CoCl 2 for 2h. For NFκB experiments, HUVECs were treated with 400 μM of CoCl 2 for 2 h with/without 0.6 μM of rMASP-1 in the last 1h. We used 1 ng/ml of IL-1β as a positive control in both CREB and NFκB experiments. Cells were then fixed in Methanol-Acetone and stained with rabbit-anti-human phospho-CREB (pCREB, 1:200) or rabbit-anti-human NFκB (1:250) followed by Alexa Fluor568-conjugated goat anti-rabbit (1:500) IgG and Hoechst 33342 (1:50000). For NFκB experiments, additional Alexa Fluor568-conjugated wheat germ agglutinin (WGA, 1:200) staining was added after for 20 min. Olympus IX-81 fluorescence microscope and an Olympus XM-10 camera were used to take photos. Photos were analyzed using CellP software (Olympus). Nuclear mean red fluorescence (pCREB) or the difference between cytoplasmic and nuclear mean red fluorescence (NFκB) were calculated.