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After removal of the culture medium, the wells were washed three times with PBS (Gibco) at 37[degrees]C, 2 mL of formalin 10% was added for fixation, and the wells were stored for 24 hours at 4[degrees]C.
A1413201, Gibco) was added to a 96-well plate and incubated at 37[degrees]C for 30 min to polymerize.
The zona pellucida of the blastocysts was removed by acid-Tyrode's solution and, the zona-free blastocysts were subsequently seeded on MEF feeder layers in a gelatin coated four well multidishes containing knockout DMEM (kDMEM; Gibco Invitrogen, USA) supplemented with 3:1 mixture of FBS (Hyclone Laboratories, USA) and knockout serum replacement (Gibco Invitrogen, USA), 1% (v/v) nonessential amino acids (NEAA; Gibco Invitrogen, USA), 0.1 mmol/L [beta]-mercaptoethanol (Gibco Invitrogen, USA), 1% (v/v) penicillinstreptomycin solution and 500 unit/mL of leukemia inhibitory factor (LIF) (Chemicon, Temecula, CA, USA), this medium was used continuously for ESCs culture [17,18].
Yeast inoculum and soluble factor of fungal metabolism: 2, 0.5 (1-5x[10.sup.6] yeast/ml) and 0.05 Mc Farland (McF) yeast inoculums in phosphate-buffered saline (PBS, Gibco Life Technologies, Grand Island, NY, USA) were incubated with three different relation between motile sperm and Candida spp.
Before use, the chondrocytes were expanded in monolayer culture for 14 days (seeding density of 5 x [10.sup.3] cells/[cm.sup.2], ~3.4 population doublings as the chondrocytes required a few days to reenter the cell cycle after being frozen) in DMEM (31885, Gibco) supplemented with FBS (10% v/v) and pen/strep (1%).
PBMCs were plated in three 6-well plates (1 x [10.sup.6] cells/well) and incubated for 3 h in RPMI1640 (Gibco) supplemented with 1% (v/v) penicillin-streptomycin and 10% FBS in an incubator at 37[degrees]C and 5% C[O.sub.2] at 100% relative humidity.
Cells at passage 3 (p3) were detached by using trypsin-EDTA 0.2% (Gibco, Carlsbad, CA, USA), fixed with methanol for 10 min at -20[degrees]C, washed with 1% BSA in PBS, and then incubated with permeabilization buffer: 0.1% Triton X-100 and 1% BSA in PBS for 10 min at 4[degrees]C.
Blood was collected from male SD rats and diluted in 1 x HBSS (Hanks balanced salt solution; Gibco) at 1: 1 ratio and layered over Percoll plus (GE Healthcare) in a centrifuge tube.
Tissue sections were then blocked for 20 minutes in PBS (Gibco) containing 5% fetal bovine serum (FBS; Gibco) and 0.1% TritonX (Sigma).
The growth medium used for C6/36 cell line consisted of 1 xminimal essential medium (MEM) containing Earle's BSS (GIBCO #11430-030), 2 mM L-glutamine (GIBCO #25030-081), 0.35 g/LNa bicarbonate (GIBCO #25080-094), 0.1 mM non-essential amino acids (GIBCO #11140-050), 1.0 mM Na pyruvate (GIBCO #11360-070), 100 units penicillin, 0.1 mg/ml streptomycin; (HiMedia Laboratories #A018-5X100ML), and 10% fetal bovine serum (GIBCO #10270-106).