Dulbecco s phosphate buffered saline dpbs

THP-1 cells (human acute monocytic leukemia cell line; KCLB No. 40202) were obtained from the Korea Cell Line Bank (KCLB, Seoul, Korea) and cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FBS, 0.05 mM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin (WelGENE). The culture was maintained at 37 °C in a humidified atmosphere of 5% CO₂. THP-1 cells were seeded at a density of 5 × 10⁵ cells/mL. The cells were differentiated into macrophage-like cells by adding 0.5 μM of PMA (Sigma-Aldrich) for 24 h prior to treatment with GO57 (link). PMA-primed THP-1 cells were washed with Dulbecco’s phosphate-buffered saline (DPBS; WelGENE) and then treated with the indicated concentrations of GOs. Prior to immunotoxicity evaluation, GO-treated cells were incubated for 6 h. For study of phagocytosis inhibition, cytochalasin D (an actin-depolymerizing agent, Sigma-Aldrich) was added to cell monolayers for 30 min prior to treatment with GOs.

All cell culture reagents were purchased from Welgene (Gyeongsan, Korea). Hep3B cells were obtained from Korean Cell Line Bank (KCLB, 88064). Hep3B cells and liver primary cells were maintained at 37 °C in a 5% CO₂ atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) (Welgene) supplemented with 5% (vol/vol) foetal bovine serum, penicillin (100 U/mol) and streptomycin (100 μg/ml). Cells were washed once with Dulbecco’s Phosphate-Buffered Saline (DPBS) (Welgene) and incubated with low glucose medium (50 mg/dl, w/o FBS; Welgene) for 18 h. P4 was treated with CD-FBS for steroid hormone delivery. All cell experiments were repeated at least three times.
For PGRMC1 overexpression, Hep3B cells were transfected with 2.5 µg of human PGRMC1 expression plasmid and Lipofectamine 2000 (Invitrogen) in Opti-MEM (Gibco) medium according to the manufacturer’s protocol. For PGRMC1 knockdown, Hep3B cells were transfected with control siRNA or PGRMC1 siRNA #1 (5′-CAGUACAGUCGCUAGUCAA-3′) and #2 (5′-CAGUUCACUUUCAAGUAUCA-U-3′) purchased from Bioneer (Daejeon, Korea).

2-Aminoethyl dihydrogen phosphate (AEP) (98%) was purchased from Tokyo Chemical Industry (TCI, Tokyo, Japan); DAEMA (98%), copper (I) bromide, 2,2-bipyridyl (99%), carboxymethyl-dextran sodium salt (10-20 kDa), titanium(IV) oxide (anatase, <25 nm particle size, 99.7%), α-bromoisobutyryl bromide (98%), mPBA, methylthiazolyldiphenyl-tetrazolium bromide (MTT), DPBF, thiol tracker violet, and 2,7-dichlorofluorescin diacetate (DCF-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and used without further purification. SCC7 and L929 cells were purchased from the American Type Culture Collection (Manassas, MD, USA). For cell culture, RPMI 1640 and fetal bovine serum (FBS) were purchased from Capricorn Scientific (Ebsdorfergrund, Germany). The antibiotic-antimycotic solution, trypsin-EDTA, and Dulbecco's phosphate-buffered saline (DPBS) were obtained from Welgene (Daegu, Korea). All experiments involving live animals were carried out in accordance with the relevant laws and institutional guidelines of Sungkyunkwan University (SKKUIACUC2019-08-22-1).

To determine the rate of apoptosis in porcine blastocysts, an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) was used for a TUNEL assay. Blastocysts were fixed in 4% (v/v) paraformaldehyde on day 6. Fixed blastocysts were washed three times with Dulbecco’s phosphate-buffered saline (DPBS; Welgene, Taipei, Taiwan) supplemented with 0.1% PBS-PVA, followed by incubation in PBS containing 1% (v/v) Triton X-100 for 1 h at room temperature for membrane permeabilization. Subsequently, the blastocysts were washed three times in PBS-PVA and stained with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5 °C. As a negative control for the TUNEL reaction, a group of blastocysts was incubated in fluorescein dUTP at 38.5 °C without terminal deoxynucleotidyl transferase. As a positive control for TUNEL reaction, blastocysts were incubated with 1000 units/mL DNase I (M0303L; New England BioLabs, Ipswich, United States) for 15 min. Subsequently, the blastocysts were washed three times in PBS-PVA and mounted on slide glasses (Marienfeld, Lauda-Königshofen, Germany) with mounting solution containing 1.5 g/mL 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA). DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope, and the numbers of apoptotic and total nuclei were counted.