Fetal calf serum (fcs)

The basic medium consisted of low-glucose Dulbecco’s modified Eagle’s medium supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin (EuroClone, S.p.A. P.IVA, Italy) and 2 mM L-glutamine (EuroClone). FCS expansion medium was made from basic medium supplemented with 10 % FCS (tested for its ability to support MSC expansion; StemCell Technologies, Vancouver, BC, Canada).
PLP was obtained by pooling four platelet units containing approximately 300 × 10⁹ platelets from the local blood bank (Finnish Red Cross Blood Service, Helsinki, Finland). Platelets were centrifuged, suspended in AB plasma (Octaplas, Finnish Red Cross Blood Service) and frozen. The platelets underwent five rapid freeze–thaw cycles. Before use, the ability of the platelet lysate to support MSC expansion was tested using FCS expansion medium as a reference. The PLP expansion medium consisted of 2.5 % AB-plasma (Octaplas), 0.5 % platelet lysate (final concentration approximately 0.8 × 10⁸ platelets/ml) and 40 IU/ml heparin (Sigma, St. Louis, MO, USA) added to the basic medium.

PBMCs were stimulated with phorbol myristate acetate and ionomycin as described below. Cells were stimulated in RPMI-1640 medium (Invitrogen) containing 10% (vol/vol) FCS (StemCell Technologies), 2 mM l-glutamine (Sigma), 100 U penicillin and 100 μg/ml streptomycin (Invitrogen) (complete medium, CM) containing 10 ng/ml phorbol 12-myristate 13-acetate (Sigma) and 1 μg/ml ionomycin (Sigma) for 4 h at 37 °C, 5% CO₂ in the presence of 20 μg/ml brefeldin A (Sigma). For spontaneous cytokine production, cells were incubated in CM in the presence of brefeldin A only.

NK-92MI cells (ATCC) were cultured in
Minimum Essential Medium α without nucleosides (Gibco), supplemented
with 12.5% total volume fetal calf serum (HyClone), 12.5% total volume
horse serum (Stem Cell Technologies), 2 mM Glutamax (Gibco), 1×
penicillin–streptomycin (Gibco), 0.02 mM folic acid (Sigma-Aldrich),
0.2 mM myo-inositol (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol
(Sigma-Aldrich) at 37°C, 5% CO₂. In addition, 50 U
recombinant human IL2 (PeproTech) was supplemented to the culture
medium during the first 5 days after starting cell culture.
Subculture of the cells was performed every 2–3 days. Viable
cell clusters were collected by centrifugation at 175g for 5 min, after which the cells were split in a 1:4 ratio to achieve
2–3 × 10⁵ cells/mL in fresh NK medium.
HT29 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco)
and 20 mM HEPES at 37 °C, 5% CO₂.

hBM-MSCs were seeded at 20,000 cells/well onto the surface of a 24 well plate. After reaching confluence, 500,000 stage II gametocytes were added to the well and co-cultured with hBM-MSCs for 48 h in α -medium (Invitrogen, Carlsbad, CA, USA) with 20% fetal calf serum (StemCell) at 37°C in 5% CO₂ atmosphere. The derived conditioned medium was collected and used for the tube formation assay. HBMEC-60 were seeded at 20,000 cells/well onto the surface of polymerized Corning Matrigel Matrix (10 mg/ml, Corning), inside a transwell support (0.4 μm pore size, Corning), using endothelial cell growth medium (EBM-2 BulletKit, Lonza). Conditioned media were added to a multiple well plate well, subsequently adding the transwell insert with polymerized Matrigel. Endothelial cells were seeded into the transwell insert, adding EBM-2 medium. The plate was incubated for 12 h to allow formation of capillary-like structures. Phase-contrast images were captured by using an inverted light microscope (Evos, Invitrogen) at 4 × magnification. Images were analyzed and quantified by using the image J analysis software (angiogenesis analyzer script) (Guidolin et al., 2004 (link)). The readouts of the image analysis software include tubule and net characteristics. Data were shown as means ± standard errors.