Giemsa stain solution

We compared the behaviors between males and females and also between females in different phases of the estrous cycle, as it is well known that both human and rodent females alter their behavior depending on the estrous cycle phase [21 (link)–24 (link)].
For all female mice, estrous cycle phase was determined by vaginal smear cytology analyses during the 4 days prior to handling. Briefly, we rinsed the vagina with 150–200 μL sterile water. The smear was placed on a glass slide (FRC-01, Matsunami Glass industries, Osaka, Japan). After drying, 50 μL of Giemsa stain solution (Merck, Tokyo, Japan) was applied to the smear, which was left to stand for 10–20 min and then washed with distilled water. After drying, the smear was observed under a light microscope (Nikon Diaphot 300, Nikon Corporation, Tokyo, Japan), and we then classified it as either proestrus, estrus, diestrus [25 (link)], or ‘not determined’ (nd) depending on the results of the analysis. To control for any behavioral effects of this procedure between sexes, males were also treated in the same way, with sterile water applied under the scrotum.

Between 500 and 10,000 cells were seeded per well in 6-well plates (VWR, Leicestershire, UK) and kept in culture under normal cell culture conditions for 5 h. After immediate attachment, cells were treated with sonicated NAs(-cisPt) and cisplatin as described before. After 7 to 10 days, colonies were washed twice with PBS, fixated in 100% ethanol (Merck, #1009832500, Darmstadt, Germany) for 15 min, stained with 10% Giemsa stain solution (Merck, Darmstadt, Germany) for 1 h and finally, cells were washed five times with deionized water. Colony formation capacity was determined by counting colonies (>50 cells) using the GelCount colony counter (Oxford Optronix, Oxford, UK), as described previously [18 (link)]. Data were obtained from at least three individual experiments in triplicates. Plating efficiency (PE) was determined by the following formula: $$\mathrm{PE}=\frac{number of treated colonies}{number of untreated colonies}$$
Subsequently, the survival fraction (SF) was calculated by the following formula: $$\mathrm{SF}=\frac{PE of treated colonies}{PE of untreated colonies}$$

Peripheral blood smears were prepared on microscope slides (Menzel-Gläser, Braunschweig, Germany) and stained with a May–Grünwald-Giemsa protocol. The staining procedure consisted of immersing the slides for 1.5 min in May–Grünwald's eosin-methylene blue solution (Merck, Darmstadt, Germany) and, after washing up the excess solution with water, staining the slides for 20 min with 10% Giemsa stain solution (Merck, Darmstadt, Germany). Furthermore, slides were left to dry for at least 30 min (Vives Corrons et al., 2004 (link)). Slides were visualized with an Olympus Vanox-T AH-2 microscope (Tokyo, Japan), equipped with an Olympus DP73 digital camera, using a 100 × oil objective (Olympus SPlan 100PL). Digital images were assessed using CellSens Entry software (V1.7, Olympus, Tokyo, Japan).

In order to evaluate the cell toxicity of FC, V79 cells (a cell line consisting of fibroblasts derived from the lungs of male Chinese hamsters) were used to inhibit colony-formation according to the direct contact method. One mL of the cell suspension (40 cells/mL) was seeded in a 24-well culture plate and cultured for seven days. The following six groups were designed for comparison: (1) control (culture medium: MEM 10) group, (2) negative control material (plastic sheet) group, (3) positive control material (polyurethane containing 0.25% zinc (ZDBC)) group, (4) experimental (fish collagen gel (for gelation: 37°C, 30 minutes)) group, (5) control (DMSO: solvent for ZDBC) group, and (6) positive control (ZDBC) group. After seven days of culture, the culture medium was removed. The cells were fixed with 1 mL of 100% methanol for five minutes and were stained with 4% Giemsa stain solution (Merck KGaA, Darmstadt, Germany). The mean number of colonies (n = 4) was counted by the naked eye and was converted to a percentage of the total number of colonies (100%) in the control group. Cell toxicity was defined as a colony-formation ratio below 30% in the FC group.

Blood smears were screened for their cellular compositions. Samples were fixed on microscope slides using methanol and stained following immersion in a standard Giemsa stain solution (Merck KGaA, Darmstadt, Germany) for 20 minutes. The cellular composition was determined after classifying 100 immune cells per individual under a 100x magnification with an Axioscope microscope (Zeiss, Oberkochen, Germany) in a blind manner to avoid bias. Immune cells included macrophages/monocytes, lymphocytes, neutrophils and eosinophils.

Chitosan (200–600 mPa·s; 0.5% in 0.5% acetic acid at 20 °C, Tokyo Kasei Kogyo, Tokyo, Japan), genipin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), acetic acid (FUJIFILM Wako Pure Chemical Corporation, Japan), disodium hydrogen phosphate (Na₂HPO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), sodium dihydrogen phosphate (NaH₂PO₄; FUJIFILM Wako Pure Chemical Corporation, Japan), Giemsa Stain Solution (Merck, Darmstadt, Germany), 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Corporation, Japan), Dulbecco’s PBS (PBS; Sigma-Aldrich, St. Louis, MO, USA), polyetherurethane (PU) film containing 0.1% zinc diethyldithiocarbamate (ZDEC) (ZDEC-PU; Hatano Research Institute, Hadano, Japan), PU film containing 0.25% zinc dibuthyldithiocarbamate (ZDBC) (ZDBC-PU; Hatano Research Institute, Hadano, Japan), high-density polyethylene sheet (HDPE; Hatano Research Institute, Hadano, Japan), Chinese hamster lung fibroblast V79 cells (V79 cells; RIKEN RCB0008 and Japan Health Sciences Foundation JCRB0603), Eagle’s minimum essential medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), fetal bovine serum (FBS; Corning Cellgro, Manassas, VA, USA), 0.5% and 0.25% trypsin solution (FUJIFILM Wako Pure Chemical Corporation, Japan and Thermo Scientific, Carlsbad, CA, USA, respectively), and CELL COUNTING KIT-8 (Dojindo Laboratories, Kumamoto, Japan) were used in this study.