Hemacolor kit

A Pappenheim staining was performed on blood, bone marrow, and peritoneal fluid harvested 1 h, 3 h, 6 h, and 12 h after CASP surgery. Therefore, a smear slide was made, and staining was carried out using the Hemacolor kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. The number and morphology of neutrophil granulocytes were determined in four high-power fields (HPFs, 400× magnification) using a light microscope (Axioskop 2, Zeiss, Jena, Germany). Rod and segmented neutrophils were counted. The mean ± SEM was calculated.

Lower wells of a modified Boyden Chamber (Receptor Technologies, Royal Leamington Spa, Great Britain) were filled with 30.2 μL serum-reduced medium with or without 10 μM Clt and separated from upper wells by a polycarbonate membrane with 8 μm pores (Osmonics, Moers, Germany). For adjustment of pH the chamber was transferred into an incubator for 30 min.
Confluent IEC-18 were starved in serum-reduced medium for 24 h with or without 100 ng/mL IFN-γ, washed with PBS and collected with Trypsin/EDTA (PAA). After centrifugation cells were resuspended in serum-reduced medium at a concentration of 200000 cells/mL and 50 μL of the suspension were pipetted in the upper compartment of the prepared Boyden Chamber.
After a migration period of six hours, the upper surface of the membrane was washed from attached cells with PBS and cells having migrated to the lower surface were fixed and stained with a Hemacolor kit (Merck, Darmstadt, Germany).
For cell counting, membranes mounted on a slide were placed under a microscope (Leica) and migrated cells in the center of the imprint of each well were counted at 200-fold magnification.

Freshly isolated ADRCs were plated in six-well plate at low density (100 cells/cm²) in DMEM/F12 containing 10% fetal bovine serum (FBS). After 12–14 days, cells were rinsed with PBS, fixed with formalin, and stained with hematoxylin solution (Hemacolor kit; EMD Millipore, Billerica, MA, USA). Colonies containing > 50 fibroblast colony-forming units (CFU-F) were counted. CFU-F frequency was calculated by dividing the number of colonies by the number of seeded cells.

To validate gene expression and gene knockdown efficiency, mosquitoes fed on uninfected and P. berghei -infected mice were obtained. Female CD1 mice were intraperitoneally inoculated with 10⁷ 
P. berghei ANKA parasitized red blood cells. The levels of parasitaemia were measured from blood smears of the mouse tail using light microscopy after staining with Hemacolor^® kit (EMD Millipore, Germany). Mice were used when the parasitaemia reached about 10% and exflagellation was observed (4–6 exflagellations/field).
Three hundred female mosquitoes per group were allowed to feed on anesthetized mice, and only fully engorged females were kept at 19–21 °C and 80% humidity.

One hundred and fifty mussels were sampled in June and September/October from each studied area and checked for the presence of Marteilia spp. by cytological analysis. A little piece of the digestive gland was used for tissue imprints. The imprints were air-dried, stained with Hemacolor Kit (Merck), and observed by microscopy 1000× using a Nikon Optiphot-2 microscope [6 ].

BAL was taken in anaesthetized mice, by instilling and withdrawing 0.5 ml of saline solution (0.9% NaCl, 2.6 mM EDTA) in the trachea. After six times lavages, BAL fluid was pooled and centrifuged, and cell number was counted using a Neubauer hemocytometer. 200 μl of BAL fluids with 2.5 × 10⁵ cells/ml were used to prepare cytospin slides, which were then stained with Hemacolor kit (Merck, Cat No. 1116740001) to identify macrophages, lymphocytes, neutrophils and eosinophils. After counting for each cell type to obtain their frequencies, number of each cell type was calculated according to the total BAL cell numbers.