Coolsnap hq

Human Embryonic Kidney 293 cells stably expressing B₂R-GFP at the level of their plasma membrane exhibit BK-induced endosomal internalization of the fluorescent receptor, maximal 30 min after stimulation but with gradual recycling to the cell surface in 1–3 h (Bachvarov et al., 2001 (link); Charest-Morin et al., 2013b (link)). A microscopic assay of extended BK analogs was based on this system and on the fact that the culture medium containing 10% heat-inactivated fetal bovine serum contains the active soluble form of ACE that remains the main BK inactivation pathway in the system (Bachvarov et al., 2001 (link)). Arg-CP(s) sensitive to Plummer’s inhibitor are also found in human plasma and inflammatory synovial fluid that contains serum proteins (Chercuitte et al., 1987 (link)), suggesting that serum-containing culture medium may contain this enzymatic activity. HEK 293 cells stably expressing B₂R-GFP were observed in epifluorescence microscopy at a 1000× magnification and photographed using an Olympus BX51 microscope coupled to a CoolSnap HQ digital camera (filters for GFP and fluorescein: excitation 460–500 nm, emission 510–560 nm, objective lens 100× oil UPlanApo, Olympus). The experiments were based on BK or BK analog stimulation of different durations to monitor either the endocytosis of B₂R-GFP (30 min) or their recycling (3 h), based on previous time course findings.

Images were acquired using a DeltaVision RT system (Applied Precision) with a Photometrics CoolSnapHQ camera using a 100× 1.40 NA UPlanSApo objective (Olympus). Images were processed for contrast enhancement and background noise reduction using ImageJ (National Institutes of Health). All image analysis was performed using custom ImageJ macros (see Source code 1). All figures and statistical analyses were generated in R. Reported p-values are the results of a two-tailed t-test.

SEM was used to examine strains grown on YPD or MYM agar for up to 14 days. Samples were prepared and visualized using a TEMSCAN LSU scanning electron microscopy as described previously (Haiser et al., 2009 (link)). To monitor the rate of exploratory growth (Video 1), an Olympus SZX12 Sterioscope and CoolSNAP HQ photometric camera were used to capture 70 frames of growth over the course of 17 hr.

Live-cell imaging was performed on a custom-built upright widefield DeltaVision microscope (Applied Precision, Olympus IX70 with a Roper CoolSnap HQ) with water-immersion objectives (Olympus). Fixed material imaging was performed on a widefield DeltaVision microscope (Applied Precision, Olympus IX70 with a Photometrics EMCCD;Olympus objectives, 1.512 oil) except for larval central nervous systems which were image at on a confocal microscope system (Fluoview FV1000 IX81; Olympus) using a 60×/1.35 NA oil objective and FV1000 software (Olympus). Images are single confocal slices, or maximum or mean intensity projections of 25 z-stacks across 5 µm depth as indicated. All images were deconvolved using softWoRx (Applied Precision) (Parton and Davis, 2006 ) in order to re-assign out-of-focus light to the point of origin.

HeLa cells were transfected with siRNA (72 hr total) followed by CFP-tagged rescue constructs 24 hr before fixation or transfected with CFP-tagged rescue constructs (24 hr) and then incubated with nocodazole (5 µg/ml, 1 hr, 37°C). Rescue constructs were made insensitive to siRNA by replacing seven nucleotides while retaining coding identity (GACCTCATGGACTGTGGAAAT to GATTTAATGGATTGCGGTAAC). To observe Golgi morphology, cells were fixed with paraformaldehyde (3.7%, RT, 15 min) and permeabilized with 0.1% Triton X-100. Cells were stained with chicken anti-GFP (Abcam) and mouse anti-p115 (mouse ascites) antibodies and visualized using an Olympus IX70 microscope with a 40× 1.35 NA Apochromat oil immersion lens (Olympus) and a charge-coupled device camera (CoolSNAP HQ). Maximum intensity projections were generated using softWoRx 4.1.0 software (Applied Precision). Cells and Golgi were segmented using CellProfiler (Carpenter et al., 2006 (link)). Golgi complex morphology was scored for each cell as the percent of p115-positive structures defined as ‘large’ (4.11 µm² for siRNA and 2.74 µm² for nocodazole-treated cells) and normalized by the mean KIF1C intensity using Matlab (Mathworks, Natick, MA; https://github.com/lee-ohlson-pfeffer/kif_golgi_fragmentation). The two-sample t-test was used to determine statistical significance.

The cells (bEnd.3, C8-D1A, and C8-B4) cultured on collagenated glass coverslips (10,000 cells/coverslip) were washed twice with PBS (pH 7.2), and fixed in PBS containing 1% PFA, for 10 minutes at room temperature. After fixation, cells were washed in PBS and permeabilized with 0.2% Triton-X-100 in PBS for 10 minutes at room temperature. The cells were then incubated with 10% BSA in 0.1% Triton-X-100 in PBS for 1 hour at room temperature to reduce nonspecific binding. After 3 washes in PBS, primary antibody against NHE1 (Abcam, ab67313, 1:200 in blocking buffer) were applied overnight at 4°C. Followed by 5 washes in PBS, AlexaFlour^TM488 secondary antibody (ThermoScientific, 1:500 in blocking buffer) was used for 1 hour at room temperature. After several washes in PBS, the coverslips were mounted to slides with Prolong Gold Antifade with DAPI (ThermoScientific, P36941) mounting media and allowed to dry. The slides were visualized using Olympus microscope (BX61), and images digitally captured (CoolSNAP HQ; Olympus).