Cell culture techniques

2.2.1.1. Cell lines

2.2.1.1.1. Macrophages like cell lines

RAW 264.7, J774A.1, and Pu5-1.8 were cultured in general cell culture medium (see 2.1.11.3.). The cells were incubated in incubators with 95% humidity and 5% CO2 at 37° C. The cells grow as adherent monolayer cel ls, and were split twice per week by mechanically scraping the cells using a rubber cell scraper before full confluently grow. Some morphological changes were observed after multiple passages for RAW 264.7 cells. These were associated with a different expression pattern of the histamine receptors (mainly H4R). Therefore, those cells were replaced every 8-10 weeks with a new aliquot from liquid nitrogen-frozen cells.

2.2.1.1.2. Supernatant isolation of L-929 and X6310 cells

The supernatants of L-929 and X6310 were used as a source of M-CSF and GM-CSF, respectively. The cells grow as adherent monolayer cells with the general cell culture medium. The cells were split twice per week and the supernatant medium was collected from the 3-4 day old cultures and filtered with 0.2 µm sterile filter, and stored at -20° C to be used as a supplementary medi um in BMDM and BMDC medium.

2.2.1.1.3. Other mouse cell lines

WEHI 164H, LA-4, and P815 were cultured with the general cell culture medium. The non-adherent cells P815 were split twice per week, and kept below 1x10⁶ cells/ml, whereas the adherent WEHI 164H and LA-4 cells were split twice per

2. Materials and methods 33 week before fully confluent grow by mechanically scraping the cells using a rubber scraper.

2.2.1.2. Mouse primary cell isolation and in-vitro generated cells 2.2.1.2.1. BMDM differentiation

The protocol used to generate BMDM was obtained from several reports with minor modifications [131;132]. In order to isolate bone marrow cells from C57BL/6 or H4R ^-/- mice were euthanized by cervical dislocation. The tibias and femurs were removed, cleaned from the muscle with a napkin, and incubated in Petri dishes containing ice-cold PBS. The bones were then sterilized by short incubation for 15 seconds in 70% ethanol, and then moved to sterile chilled PBS. Bone marrow cells were obtained by cutting the ends of the bones under sterile conditions, and flushing the bone marrow cells out with chilled PBS using a 27G needle, the cells were counted with Türk’s solution to exclude the erythrocytes, and 10x10⁶ cells were seeded in 20 ml BMDM medium (see 2.1.11.1.) in 10 x 10 cm Sterilin square dishes, and incubated in humid incubator at 37° C with 5% C O2 for 7 days. On day 5, the non-adherent cells were discarded with the old medium and replaced by fresh 20 ml BMDM medium. BMDM cells were also generated in 6-well plates similar to the previous protocol, but bone marrow cells were seeded at 1x10⁶ cells in 4 ml BMDM medium. Macrophage markers were analyzed on day 7 by FACS to ensure the successful differentiation (see 2.2.3.1.).

2.2.1.2.2. Isolation of mouse peritoneal macrophages

In order to enhance yield of peritoneal exudates cells (PEC) and macrophages harvested from the peritoneal cavity, sub-inflammation induction prior to the harvest is essential. Such inflammatory induction was generated by injecting 1 ml sterile PBS with a 25 G needle in the peritoneal cavity; 14 hours later the mouse was euthanized by decapitation. After sterilizing the abdomen with 70% ethanol, 10 ml of chilled PBS containing 10% FCS were used for peritoneal lavage, the procedure was to use 20 ml syringe attached to 19 G needle, and inject the medium in the intact peritoneal cavity, with the beveled end of needle facing up without hitting the intestine. After gently removing it, the same needle was reinserted in the upper part of the

2. Materials and methods 34 peritoneum, with the beveled end of needle facing down, and the peritoneal fluid was withdrawn slowly. The peritoneal exudates cells were amounted to be around 2x10⁶ cells per mouse, and were seeded at 1x10⁶ in 60 mm cell culture dishes for 2 hours to ensure macrophages adherence. Non-adherent cells were discarded, and only adherent cells were used for RNA isolation or phagocytosis assays. The samples with PEC above 2x10⁶ were discarded because this might indicate a bacterial infection in the peritoneal cavity.

2.2.1.2.3. BMDC differentiation

Bone marrow cells were obtained as mentioned before (see BMDM differentiation). 2x10⁶ cells were seeded in 10 ml BMDC medium (see 2.1.11.2.) in 10 cm Petri dishes, or 2x10⁵ cells in 1 ml BMDC medium in 24 well plate. On day three the cells were supplied with additional (10 or 1) ml fresh medium, respectively. On day six 10 or 1 ml respectively from the supernatant were centrifuged and the pellet was resuspended in a fresh (10 or 1) ml medium and added to the original dish. On day eight, the same step as in day six was repeated. The cells on day 8 were considered as immature DC which express CD11c, the specific mouse DC marker.

The cells could be incubated for longer time up to day 10 when new medium is frequently supplied. BMDC were detached from the dishes by vigorously aspiring the medium with the pipette [133].

2.2.1.2.4. Spleen and lymph-nodes cells

C57BL/6 mice were euthanized by cervical dislocation. Spleen, axillary and brachial lymph-nodes were immediately isolated and incubated on chilled PBS.

Organs homogenization was performed by pressing them through a cellular strainer (40 µm) with a syringe plunger. The cells and the strainer were washed with 10 ml of chilled PBS and resuspended to ensure the cell separation and formation of single cell suspension. Afterwards, spleen and lymph node cells were used for RNA isolation, or stained by specific antibodies for FACS experiments (spleen only).

2. Materials and methods 35 2.2.1.2.5. Spleen macrophage preparation

In order to study the macrophages residing in spleen, the spleen single cell suspension was prepared and the cells were left for 2 hours in 10 cm Petri dishes to adhere. After that, the non-adherent cells were washed away, and the adherent cells were washed by PBS and gently scraped by rubber scraper and analyzed by FACS, after staining the cellular markers with labeled-antibodies (see 2.2.3.1.).

2.2.1.3. Cell stimulation, maturation and activation 2.2.1.3.1. Classical and alternative activation of BMDM

In order to generate M1 macrophages by classical activation, LPS (E. coli) was used at a concentration of 100 ng/ml for 24 h, or IFN-γ at a concentration of 50 IU/ml for 24 h. The activation was performed with BMDM generated in 6 wells plates.

BMDM alternative activation to generate M2 macrophages was performed by incubating BMDM with IL-4 for 24 h at a concentration of 50 ng/ml. M1 and M2 macrophages were later subjected to RNA isolation and real-time PCR experiments to quantify the mH4R expression.

2.2.1.3.2. RAW 264.7 cells stimulation

RAW 264.7 cells were seeded in 6 well plates for 24 hours at a concentration of 2 x 10⁵ in 3 ml medium. Later, medium was substituted with medium alone or medium containing IFN-α (120 IU/ml), IFN-γ (50 IU/ml), and TNF (50 ng/ml) separately for 3, 6 and 24 hours. At each time point, the stimulated and non-stimulated cells were harvested to isolate RNA for real-time PCR.

2.2.1.3.3. BMDC maturation

BMDC were incubated on day 8 with 100 ng/ml LPS for 24 hours in order to induce the maturation. The maturation was examined by FACS to inspect the expression of CD86 and MHC II (mature DC markers) among the CD11c positive population.

2. Materials and methods 36 2.2.1.3.4. BMDC maturation with histamine and 4MEH in the presence or absence of LPS

BMDC generated in 24 well plates were incubated on day 8 with 10 µM histamine or 10 µM 4MEH. As positive control, cells were incubated with 10 ng/ml, or 50 ng/ml LPS alone or with histamine and 4MEH separately (10 µM). As negative control, cells were incubated with 10 µl/well sterile H2O. The experiment was performed in duplicate for each incubation condition, and the cells were analyzed by FACS on the next day for maturation markers (CD86 and MHC class II).