Vybrant cm dii cell labeling solution

HeLa cells were seeded (at a density of 1.5 × 10⁵ cells/dish) in 35 mm glass bottom dish (AGC Techno Glass) and incubated for 24 h. After cell condition became stable, the cells were incubated with 0.1 µM of each peptide and 1% DMSO at 37 °C for 4 h. The cells were washed with phosphate buffered saline (PBS, FUJIFILM Wako Pure Chemical) three times and cell membrane was stained with 5 µM of Vybrant™ CM-DiI Cell-Labeling Solution (Thermo Fischer Scientific) for 5 min. The cells were washed with PBS and fixed with 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical) for 10 min. After washing with PBS three times, the cells were mounted with Prolong Diamond Antifade mountant with DAPI (Thermo Fischer Scientific). Intracellular distribution of green fluorescence from the peptide was observed using Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany) at the Support Unit for Bio-Material Analysis in RIKEN Center for Brain Science, Research Resources Division. The fluorescence intensity was adjusted with Leica LAS X software to compare the fluorescence distribution from peptides and cell membrane.

Hemocyte attachment to mosquito midguts in response to rTNF-α treatment was examined by immunofluorescence analysis as previously described [18 (link)] with slight modification. Two days after treatment with either rTNF-α or 1XPBS, mosquitoes were injected with 69nl of 100μM Vybrant CM-DiI cell labeling solution (ThermoFisher) and allowed to recover for 30min at 27oC. Mosquitoes were injected with 200nl of 16% paraformaldehyde (PFA), then the entire mosquito was immediately submerged/ incubated in a solution of 4% PFA for 40 sec prior to transfer in ice-cold 1XPBS for midgut dissection. Dissected midguts were incubated overnight in 4% PFA at 4oC for fixation. The following day, midguts were washed with ice-cold 1XPBS three times and permeabilized with 0.1% TritonX-100 for 10 minutes at room temperature. After washing three times with 1XPBS, tissues were blocked with 1% BSA in 1XPBS for 40 minutes at room temperature and stained with Phalloidin-iFluor 405 Reagent (1:400 in PBS; abcam, ab176752) for 1 hour to visualize actin filaments. Midguts were washed with 1XPBS to remove excess staining, placed on microscope slides and mounted with ProLong Diamond Antifade Mountant (ThermoFisher). Samples were imaged by fluorescence microscopy using a Zeiss Axio Imager 2 and analyzed to determine the number of hemocytes attached to individual midguts from each experimental condition.

To investigate the recruitment of hemocytes to the basal laminal of the midgut of mosquitoes infected with the CSP_mut, 3–5-day-old A. stephensi were injected with 69 nL of 100 µM Vybrant^® CM-DiI cell labeling solution (Thermo Fisher Scientific), the day prior to gene-silencing experiments, as previously described^{5 (link)}. To maintain the attachment of hemocytes to the midgut, 276 nL of 16% paraformaldehyde was injected into the anesthetized mosquitoes. The mosquitoes were allowed to stand for 40 s, and then the midgut was dissected in a 4% paraformaldehyde solution, and further fixed with 4% paraformaldehyde overnight at 4 °C. The following day, the midguts were washed twice with PBS, blocked with PBS containing 1% BSA for 40 min, and washed twice with the same solution. For actin and nuclei staining, midguts were incubated for 30 min at room temperature with 1 U of Alexa Fluor^® 647 phalloidin (Thermo Fisher Scientific), and DAPI (Beyotime Biotech). Tissues were mounted on microscope slides using Dako Fluorescence Mounting Medium (Agilent). Hemocytes were visualized by confocal microscopy (Leica TCS SP8) and LAS AF Lite was used for image acquisition and export, the number of hemocytes per midgut in each biological condition was analyzed.

To examine the persistence of bone marrow-derived MSC in the hindlimb ischemia model in BALB/c nude mice, the cells were stained with Vybrant™ CM-DiI Cell-Labeling Solution (Thermo Fisher Scientific, Waltham, USA) as described before [22 (link)], prior to injecting the cells into the animals. Five million viable cells were resuspended in 50 μl PlasmaLyte A and injected i.m in both ischemic and normal limbs. Evaluation of signal intensity from CM-DiI-labeled cells was performed after cell injection on days 1, 3, 6, 11, 14, 21, and 28 using the In Vivo Imaging System (IVIS) Xtreme Imaging System (Bruker, Massachusetts, USA). The images were quantified using Carestream molecular imaging software. As per the IVIS Xtreme Imaging System guidelines, all the animals were imaged before the cell injection to normalize autofluorescence. Average pixel intensity from each group was calculated from the individual animal’s pixel area intensity.

Hemocytes were stained in vivo using Vybrant CM-DiI Cell-Labeling Solution (Invitrogen) as we have previously described^{9 (link)}. Briefly, live mosquitoes were injected with ~ 0.4 μL of a solution consisting of 67 μM CM-DiI and 1.08 mM Hoechst 33342 (nuclear stain; Invitrogen) in PBS. Mosquitoes were incubated in the environmental chamber for 20 min, and then fixed by injecting 16% paraformaldehyde into the hemocoel. Ten minutes later, a razor blade was used to separate the abdomen from the head and thorax, and to bisect the abdomen along a coronal plane such that the dorsal and ventral sides were separated. The dorsal abdomens were then immersed in PBS containing 0.1% Triton X-100 and the internal organs were removed. The dorsal abdomens—containing the heart, periostial hemocytes and pericardial cells—were rinsed briefly in PBS and mounted between a glass slide and a coverslip using Aqua-Poly/Mount (Polysciences; Warrington, PA, USA).

Haemocytes were labelled with the Vybrant CM-DiI Cell-Labeling Solution (Invitrogen) as we described [5 (link)]. Briefly, live mosquitoes were injected approximately 0.4 µl of 67 µM CM-DiI and 1.08 mM Hoechst 33342 (nuclear stain; Invitrogen) in PBS, incubated at 27°C for 20 min, and injected 16% paraformaldehyde. Ten min later, abdomens were bisected along a coronal plane, and the dorsal portions containing the heart and periostial haemocytes were mounted on glass slides using Aqua-Poly/Mount (Polysciences; Warrington, PA, USA). CM-DiI stains live haemocytes, and we have used this technique to monitor haemocyte location, number and migration, as well as haemocyte-mediated phagocytosis and melanization [4 (link)–9 (link),13 (link)–15 (link),40 (link)].

For in vivo hemocyte staining we used Vybrant™ CM-DiI Cell-Labeling Solution (Invitrogen™) essentially as described (29 (link)). Briefly, female mosquitoes were placed on petri dish on ice and injected with 150 nanoliters containing 100 μM CM-DiI, freshly prepared in sterile water, after blood or sugar meal at specific time points. Injections were done using a nano-injector Nanoject III (Drummond Scientific Company). After injections, mosquitoes are placed on cages at 28°C until specific time points for midguts dissections.