Protocol workflow (Figure 1)
Isolation and culture of monocytes:
1. Dilute 10 mL of peripheral blood 1:1 in PBS, close the bottle and mix inverting them carefully.
2. Place 8 mL of Ficoll-Paque Plus in 50 mL conical tubes.
3. Cover the Ficoll-Paque with the blood + PBS mixture using a serological pipette as slowly as possible to avoid mixing the Ficoll-Paque and the blood + PBS mixture.
4. Centrifuge the sample at 2,500 rpm and 20 °C for 20 min. The rotor brakes have to be fully deactivated to effectively prevent phase mixing. It is also very important to take into account that temperature differences could change the density rates of the liquids and can have a negative impact on the separation results.
5. Carefully aspirate the middle white layer containing the PBMCs. During this step try to transfer as little Ficoll-Paque as possible.
6. Wash PBMCs two times with PBS 1X (5 min 1500 rpm) and add 20 ml of complete medium.
7. Pre-treated 6-well plates with 2 ml of porcine serum for 1 h at 37 ºC to facilitate adherence.
8. Aspirate the serum and dry completely the plate before culturing 2 ml of PBMCs in RPMI for at least 3 h.
9. After that, wash away the non-adherent cells washing 3 times with fresh RPMI, and add 2 ml of complete RPMI to culture adherent cells.
Monocytes-derived macrophages differentiation and polarization:
1. After 24 h of culture, replace the medium and add 2 ml of M1 differentiation medium or M2 differentiation medium for M1 or M2 differentiation, respectively. For M0 control, add 2 ml of complete RPMI.
2. On day 7 of culture, replace the medium and add 2 ml of M1 polarization medium or M2 polarization medium for M1 or M2 polarization, respectively. For M0 control, add 2 ml of complete RPMI.
3. After, 24 h of culture with polarization medium, observe the cells under the optical microscope. M1- and M2- macrophages, will present morphological differences.
1. Aspirate and discard the culture medium, and rinse with PBS. Detach porcine macrophages from culture flasks with PSB-EDTA 5mM pH 7 for 10 min at 37ºC and suspended them in PBS containing 2% FBS.
2. Incubate 2 × 105 cells for 30 min at 4 °C with appropriate concentrations of the monoclonal antibodies: CD14, CD16, CD163, CD206, and SLA-II (Swine Leukocyte Antigen class II).
3. Wash cells and resuspend in PBS containing 2% FBS.
4. Flow cytometric analysis is performed on a FACScalibur cytometer (BD Biosciences) after the acquisition of 10.000 events. Cells are primarily selected using forward and side scatter characteristics and fluorescence is analyzed using CellQuest software (BD Biosciences).
5. Isotype-matched negative control antibodies should be used in all the experiments.
6. Calculate the % of positive cells for the different markers and the mean relative fluorescence intensity by dividing the mean fluorescent intensity (MFI) by the MFI of its negative control.
1. Aspirate and discard the culture medium, and rinse with PBS. Detach porcine macrophages from culture flasks with PSB-EDTA 5mM pH 7 for 10 min at 37ºC.
2. For isolation RNA, use the PureLink RNA Mini Kit (ThermoFisher Scientific), according to the manufacturer’s instructions.
3. Add 300-600 μL, depending on cell number, of Lysis Buffer with 10% of 2-mercaptoethanol to the cell pellet and transfer the mix to a RNase-free tube. Add one volume 70% ethanol to each volume of cell homogenate and transfer the sample to the Spin Cartridge. Consecutive centrifugations and ethanol washes are performed. Finally, resuspend the pellet in DEPC-treated water.
4. Measure RNA quality and concentration using a UV spectrophotometer, such as NanoPhotometer Implen GMBH NP80 (Biotek, Winooski, VT, USA). Only the RNA samples with a 260/280 nm absorbance ratio between 1.8 and 2.1 and with a 260/230 mm absorbance ratio greater than 2.0 should be retrotranscribed to complementary DNA (cDNA) and amplified by PCR.
5. Synthesize cDNA from 100fg-1ug of RNA in reverse transcription reaction using iScript™ Reverse Transcription, according to manufacturer's instructions.
6. Prepare amplification reactions in triplicate with adequate concentrations of primers, cDNA, DEPC water, and Taq DNA Polymerase Recombinant kit (Invitrogen), according to manufacturer’s instructions. Amplify in a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc.)
7. Gene expression levels are analyzed and normalized with the Thermo Fisher Cloud software (also called Thermo Fisher Connet) using hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference gene.