Axiocam erc

Lacerates were surgically removed from both symbiotic and aposymbiotic adult Aiptasia and collected as described above. Development was tracked by recording the daily number of tentacles of each individual lacerate for 20 days. The mean number of tentacles per day was calculated for both symbiotic and aposymbiotic lacerates. P-values for comparisons between the number of daily tentacles for both groups were calculated using a Mann–Whitney U test, since the data were not assumed to follow a normal distribution. Data were visualized with RStudio (RStudio Team, 2021 ).
A new batch of lacerates was used to track tentacle and mesentery patterning during development. We established a series of developmental stages reflecting major tentacle patterning events that were consistently observed in Apo and Sym lacerates. Brightfield images were obtained with a Leica M165 FC stereo microscope and Axiocam ERc (ZEISS) and edited for brightness and contrast with FIJI (Schindelin et al., 2012 (link)) and Photoshop (Adobe).

To culture MCF-7, MCF-7/Fulv, and MCF-7/Tam cells in three-dimensional (3-D) conditions, a soft agar assay was applied using a standard procedure. Briefly, in a 12-well plate, a bottom layer consisting of 0.7% agar in 1 mL rf-RPMI with 10% CSS was first allowed to solidify in each well. The appropriate number of cells (75 × 10³ cells/well) was mixed with 1 mL rf-RPMI with 10% CSS that contains 0.5% agar and applied on the top of the bottom agar media layer. Each well was further supplemented with 1 mL fresh rf-RPMI with 10% CSS once a week. After 10–15 days of incubation at 37°C, 5% CO₂, and 100% humidity, cells were stained with crystal violet and visualized in an inverted microscope (Axiovert 40 CFL, AxioCam ERc, Zeiss, Germany) at a magnitude of ×4 or ×10.

A subset of parasitoid females that emerged within 24 h from the treatments ‘Tb’ and ‘Ot’ (egg mass size = 20 eggs), was randomly selected to investigate the host handling time, i.e. the time needed for the wasps to complete an oviposition bout. These wasps were offered an egg mass inside a small Petri dish (4 cm diameter) and the host handling time was scored for the first successful parasitism event. A video camera (Zeiss Axiocam ERC) mounted on a stereoscope (Zeiss Stereo Discovery.V12) was used to record the wasp behavior with the aid of AxioVision SE64 Rel. 4.9.1 software for image acquisition and analysis. The time taken by T. basalis and O. telenomicida to perform the following behavioral steps during an oviposition bout was recorded: (i) tapping the host egg with the antennae (= drumming), (ii) puncturing the host chorion with the ovipositor (= drilling), (iii) withdrawing the ovipositor while staying motionless on the host egg (= resting), (iv) drinking droplets of ooplasm oozing from the punctured host egg (= host‐feeding), (v) inserting the ovipositor to lay egg inside the host (= oviposition), and (vi) sweeping the ovipositor on the surface of the host egg (= marking). For both parasitoid species 10 replicates were carried out.

Cells were suspended in 1 mL of 10% FBS DMEM medium containing 0.5% agarose and plated on a semisolid medium (DMEM with 10% FBS and 0.7% agarose) in 12-well plates (2 × 10⁴ cells/well). Cells were then placed in a 37 °C and 5% CO2 incubator. The next day, TKIs were added to the supernatant growth medium and changed every two days. The IC50 values for each drug were calculated from a log ([drug]) vs. normalized response curve fit in Excel. Colonies of cells were counted after 12 days of incubation with drugs. Images were obtained using an inverted microscope (Axiovert 40 CFL, AxioCam ERc, Zeiss). All experiments were done in triplicates. Cell colony area was calculated with ROI Manager on Image J software.