healthmedicinet

Study population

Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat preparations
derived from the whole blood of 8 healthy male donors anonymously identified by code
numbers (mean age 48.5?±?13.4 years). Buffy coats no more available for clinical use
(not used within 24 h after collection) were provided by the Transfusion Unit of the
Ospedale Maggiore (Bologna) as approved by the Centro Regionale Sangue (Prot. N.32041/10-14-01).

Mononuclear cell isolation and CD14
⁺
, CD8
⁺
, CD19
⁺
and CD4
⁺
cell purification

PBMCs were isolated by conventional density gradient centrifugation over Lympholyte-H
gradient medium (??=?1.077?±?0.001 g/cm
³
; Cedarlane, USA). Briefly, buffy coats were diluted with 3 volumes of PBS and layered
over Lympholyte-H (at a 2:1 ratio) in 50 ml conical tubes, then centrifuged at 800?×?g
for 20 min at room temperature. PBMCs collected at the interphase were washed twice
with PBS (400?×?g for 10 min), counted and resuspended in PBS containing 0.5 % fatty
acid-free bovine serum albumin (GE Healthcare, Milan, Italy) and 2 mM EDTA, pH 8.0.
PBMCs were then immediately used for purification of cellular subpopulations.

CD14
⁺
, CD19
⁺
and CD8
⁺
cellular subpopulations were purified using an AutoMACS Pro Separator (Miltenyi Biotec,
Bologna, Italy) and specific microbeads for positive selection (CD14, CD19 and CD8
Microbeads; Miltenyi Biotec, Bologna, Italy). The CD4
⁺
cell population was positively purified from the CD14 negative eluted fraction with
the same instrument and CD4 Microbeads (Miltenyi Biotec, Bologna, Italy). MACS isolated
cell subsets were collected, counted and an aliquot of those subsets was used to assess
population purity. Cells were incubated for 15 min at room temperature with FITC-conjugated
antibodies against CD14, CD19, CD8 and CD4 (Miltenyi Biotec, Bologna, Italy), washed
twice in PBS and evaluated by flow cytometry (FACSCanto II, BD). FITC fluorescence
was filtered by a 530?±?21 bandpass filter. The frequency of positive cells was measured
as the percentage of gated cells in the FITC channel with activities above 99 % of
the corresponding isotype control. Purities of the obtained cell populations were
(mean?±?SD): 91.9?±?3.7 % for CD14
⁺
, 93.8?±?6.9 % for CD19
⁺
, 92.4?±?4.4 % for CD8
⁺
and 94.9?±?3.7 % for CD4
⁺
. Freshly isolated cell populations were washed, lysed and pelleted as described below.

In vitro cultures

All cell culture media, sera and reagents were purchased from Euroclone SpA, Milan,
Italy. Immortalised leukocytic cell lines (Jurkat, Raji and THP-1 cells) were kindly
provided by Rizzoli Orthopedic Institute and University of Bologna. Jurkat, Raji and
THP-1 cells were cultured in RPMI-1640 medium containing 10 % fetal bovine serum (FBS),
L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ?g/ml). Cells were
maintained in a humidified environment at 37 °C and 5 % CO
₂
and cultured in polystyrene culture flasks. Cells were passaged every 3 days, thus
maintaining cell number between 1?×?10
⁵
and 1?×?10
⁶
per ml of medium (Jurkat cells), between 4?×?10
⁵
and 3?×?10
⁶
per ml of medium (Raji cells), or between 2?×?10
⁵
and 1?×?10
⁶
per ml of medium (THP-1 cells), according to the standard protocol provided by the
American Type Culture Collection (ATCC).

To compare freshly isolated healthy human lymphocytes with primary cultured lymphocytes,
PBMCs were isolated from the buffy coat of a 40 year old healthy male donor on a density
gradient, as described above. Cells recovered from the gradient interphase were washed
in PBS, resuspended in RPMI supplemented with 10 % FBS, counted and seeded in flasks
at a density of 2?×?10
⁶
cells/ml for 3 h to allow monocyte adherence. After this time, lymphocytes were recovered
and maintained in culture in RPMI-1640 medium supplemented with 10 % FBS for 96 h
in the absence or presence of 20 ?g/ml phytohaemagglutinin (PHA) and then recovered.
Freshly isolated and 96-h cultured lymphocytes were washed, lysed and pelleted as
described below.

Membrane isolation

Cells (7?×?10
⁶
) were collected in a 15 ml tube, centrifuged at 500?×?g for 5 min and resuspended in 10 ml of PBS. The wash was repeated five times in order
to discard traces of medium and serum used during the culture process. Cells were
then resuspended into 500 ?l of PBS and collected in a 1 ml tube, to which 500 ?l
of sterile H
₂
O were added. Cells were then centrifuged for 30 min at 15,000?×?g in a refrigerated centrifuge at 4 °C. The collected membranes were resuspended in
1 ml of PBS:H
₂
O 1:1 and washed 5 times following the same procedure.

Fatty acid composition analysis

Cell and cell membrane lipids were extracted with CHCl
₃
/MeOH 2:1 (vol/vol) and then incubated with 0.5 M KOH in methanol for 10 min at room
temperature, thus trans-esterifying fatty acids linked by ester bonds to alcohols.
The corresponding fatty acid methyl esters (FAMEs) were formed, extracted with n-hexane
and separated by gas chromatography. FAMEs were separated by gas-chromatography in
an Agilent 7820A GC System (Agilent Technologies, Santa Clara, USA) fitted with a
30 m?×?0.32 mm DB23 capillary column, film thickness 0.25 ?m, and a Flame Ionization
Detector (FID). Helium was used as carrier gas at 2.54 ml/min and the spilt injector
was used with a split ratio of 10:1. Injector temperature was 250 °C and detector
temperature was 260 °C. The column oven temperature was maintained at 50 °C for 2 min
after sample injection and was programmed for the following temperature gradient:
10 °C/min from 50 °C to 180 °C, 3 °C/min from 180 °C to 200 °C and holding at 200 °C
for 6 min. The separation was recorded with G6714AA SW EZChrom Elite Compact (Agilent
Technologies). FAMEs were identified by comparison with standards purchased from NuCheckPrep
Inc., Elysian, USA. FAMEs are expressed in weight %, based upon the % contribution
of the peak area of each FAME in the chromatogram. To take into account the different
signal of the detector for different molecules, a correction factor was applied to
the experimental data coming from the integration of the chromatograms. The total
of the peaks analysed for each chromatographic run was 100.

Fatty acid aggregates were calculated as follows:

? SFA?=?14:0?+?15:0?+?16:0?+?17:0?+?18:0?+?20:0?+?22:0?+?23:0?+?24:0;

? MUFA?=?16:1n-7?+?18:1n-9?+?18:1n-7?+?20:1n-9?+?22:1n-9?+?24:1n-9;

? PUFA?=?18:2n-6?+?18:3n-6?+?18:3n-3?+?20:3n-9?+?20:3n-6?+?20:4n-6?+?20:3n-3?+?20:5n-3?+?22:2n-6?+?22:4n-6?+?22:5n-6?+?22:5n-3?+?22:6n-3;

? trans FA?=?t16:1n-7?+?t18:1n-9;

? Omega3?=?18:3n-3?+?20:3n-3?+?20:5n-3?+?22:5n-3?+?22:6n-3;

? Omega6?=?18:2n-6?+?18:3n-6?+?20:3n-6?+?20:4n-6?+?22:2n-6?+?22:4n-6?+?22:5n-6;

? Omega7?=?16:1n-7?+?18:1n-7;

? Omega9?=?18:1n-9?+?20:1n-9?+?22:1n-9?+?24:1n-9.

Indexes were calculated as follows:

Unsaturation Index (UI)?=?? [mi*ni], where mi?=?mole percentage, ni?=?n° of double
bonds;

Peroxidability Index (PI)?=?? monoenoic*0.025?+?? dienoic?+?? trienoic*2?+?? tetraenoic*3?+??
pentaenoic*6?+?? hexaenoic*8.

Refeed® supplements

Refeed® supplements (Remembrane Srl, Imola, Italy) are a completely defined combination
of non-animal derived lipids and antioxidants (NuCheckPrep Inc., Elysian, USA; Sigma
Aldrich, St. Louis, USA; Applichem an ITW Inc., Chicago, USA) solubilised in 1 ml
of ethanol (Sigma Aldrich). 1 ml of Refeed® was diluted in 560 ml of complete cell
growth medium, the resulting ethanol concentration being??1 % (vol/vol) in the final
medium. Refeed® WT (Wild-Type), Refeed® CVD (Cardiovascular Disease) and Refeed® O3+
(Omega-3 plus) were specifically developed for Jurkat cells and their compositions
are shown in Table 1.

Table 1. Composition of supplements used in the study

Statistical analysis

Descriptive and analytical tests were performed with IBM SPSS® Statistics, Version
21.0. Data were checked for normality using the Shapiro-Wilk normality test. Normally
distributed data were compared using Student’s t-test, to determine if two sets of fatty acid data were significantly different from
each other. Non-normally distributed data were compared using the Mann–Whitney U-test. For sums and other aggregates (UI, PI), Student’s t-test was performed when the aggregate was composed of all normally distributed individual
FAMEs, while Mann–Whitney U-test was performed when the aggregate was composed of one or more not normally distributed
FAMEs. For each test, the significance threshold was P??0.05.