Lipid Droplet

To analyze their lipid droplet formation for the 3D culture, the organoids were transferred to 6 super-low attachment well dishes, incubated in 0.2% BODIPY (# D3922, Thermo Fisher Scientific) in PBS for 1 hr, and then fixed in 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. Fluorescence intensity of the BODIPY-stained lipid droplets was measured using a Nikon A1 confocal microscope (Tokyo, Japan) and quantified using Image J software version 2.0.0 (NIH, Bethesda, MD).
For immunostaining of the 3D culture, organoids prepared as above were fixed in 4% PFA/PBS overnight. After blocking in 3% BSA/0.1% PBS for 3 hrs at room temperature, organoids were washed 3 times for 30 min with PBS. Samples were incubated with a primary antibody including a rabbit anti-collagen monoclonal antibody (collagen 1; # 600-401-103-0.1, collagen 4; # 600-401-106-0.1, or collagen 6; # 600-401-108-0.1, Rockland Immuno-chemicals Inc.) or mouse anti-FN monoclonal antibody (# G0717, Santa Cruz Biotechnology) at 1:200 dilutions overnight at 4 °C. After a subsequent wash in PBS, the organoids were incubated with goat Alexa Fluor 488 anti-rabbit IgG (# A-11070, Thermo Fischer Scientific) or goat Alexa Fluor 594 anti-mouse IgG (# A-11020, Thermo Fischer Scientific) at 1:500 dilutions for 3 hrs at room temperature. Subsequently, for F-actin and nuclear staining, slides were incubated with Alexa Fluor 594 phalloidin (# 20553, Funakoshi) and DAPI (# D523, Dojindo) at 1:1000 dilutions for 3 hrs at room temperature. Slides were then mounted in Prolong Gold before observation by confocal microscope.

To stain hemocytes inside live mosquitoes, 0.2 µl of a solution consisting of 75 µM CM-DiI (Vybrant CM-DiI Cell-Labeling Solution, Invitrogen) and 0.75 mM Hoechst 33342 (Invitrogen) in PBS was injected into mosquitoes. It was crucial that this solution be injected within minutes of its preparation, as once the CM-DiI is placed in an aqueous environment its hemocyte-staining effectiveness rapidly decreases, approaching 0% after 10–15 min of mixing. After CM-DiI injection, mosquitoes were immediately returned to 27°C and 75% relative humidity for an incubation period of 20 min.
Circulating hemocytes were collected by perfusing the hemolymph onto the center of 1 cm diameter etched rings on Rite-On (Gold Seal; Portsmouth, N.H.) glass slides [25] (link). Cells were allowed to adhere to slides for 20 min at room temperature, fixed for 20 min with 4% formaldehyde in PBS, washed 3 times for 5 min with PBS, and coverslips were mounted with Aqua Poly/Mount (Polysciences; Warrington, PA). Visual examination of adherent perfused hemocytes was conducted using a Nikon 90i compound microscope (Nikon; Tokyo, Japan) equipped with a Nikon Intensilight C-HGFI fluorescence illumination unit and a CoolSNAP HQ² digital camera (Roper Scientific; Ottobrunn, Germany). Cells were counted at 1000× magnification by scanning the slides from the far left to the far right until 50 hemocytes from each individual mosquito were visualized, also keeping track of the number of fat body cells observed. Intact cells were first identified as either hemocytes or fat body by confirming the presence of a nucleus using fluorescence microscopy (Hoechst 33342) and comparing cell morphology by differential interference contrast (DIC) microscopy to the descriptions of previous authors [18] (link). Specifically, hemocytes are significantly smaller than fat body cells and do not contain large lipid droplets. Hemocytes were then examined for the presence of phagocytosed GFP-expressing E. coli, and all cell types were examined for their incorporation of CM-DiI. Three treatments were performed (naïve, LB injected and E. coli injected), and for each treatment, hemocytes from 15 individual mosquitoes that originated from 5 independent but paired cohorts were examined (i.e., for each treatment, 3 mosquitoes per cohort).

Cells undergoing osteoblast differentiation were fixed in 70% cold ethanol (5 min, − 20 ºC). After drying the wells, to reveal calcium-rich mineralisation deposits the cells were incubated at room temperature with a solution of Alizarin Red (40 mM, pH 4.2) for 20–30 min. Prior to acquiring the images, the cells were gently washed with distilled water to avoid unspecific staining. For quantitation, the staining was eluted with 10% (w/v) Cetylpyridinium solubilized in 10 mM sodium phosphate buffer (pH 7.0), and the absorbance measured at 570 nm.
Cells undergoing adipogenesis were fixed with formalin for 1 h. After washing the wells with distilled water and 60% isopropanol the wells were dried. To reveal the presence of lipid droplets the cells were stained with a 21% (w/v) solution of Oil Red O for 10 min. Prior to acquiring the images, the wells were gently washed with distilled water to avoid unspecific staining. Staining images were quantified using the image analysis software ImageJ.