Trypan blue solution

Apoptotic and necrotic cells were detected using an annexin V-FITC/Ethidium homodimer III (EtD-III) staining kit (Biotium), according to the manufacturer’s protocol. Briefly, control and infected cells were washed twice with PBS then incubated with appropriate dilutions of annexin V-FITC and EtD-III in binding buffer for 30 min. Washed twice in binding buffer and fixed with 4% PFA containing 1.25 mM calcium chloride (CaCl₂) at room temperature for 30 min, washed three times in PBS containing 1.25 mM CaCl₂ and mounted on slides using Prolong Gold antifade reagent mounting medium with DAPI. Viability of infected cells treated with U0126 and CRID-3 was assessed using Trypan blue staining, briefly, cells were washed twice with PBS and incubated with trypsin for 10 min, cell suspension was then mixed 1:1 (v/v) with 0.4% trypan blue solution (Amresco) for 5 min, and viability was assessed using LUNA Automated Cell Counter (Logo Biosystems).

Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 0.4% trypan blue solution were purchased from Amresco (Solon, OH, USA). Progesterone, insulin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2′,7′-dichlorofluorescein diacetate (DCF-DA), retinoic acid, hydrocortisone, and LPS from E. coli O111:B4 were obtained from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM/high glucose and low glucose), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Welgene (Gyeongsan, Korea). Phosphate-buffered saline (PBS) and trypsin were obtained from Gibco (Grand Island, NY, USA). Primary antibodies against COX-2, NF-κB p65, proliferating cell nuclear antigen (PCNA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and secondary antibodies against goat anti-rabbit IgG-HRP and donkey anti-goat IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRIzol reagent were obtained from Life Technologies (Eugene, OR, USA).

Polyethyleneimine 25 KDa (PEI 25 KDa), 1, 4-Butanediol bis (chloroformate), Dimethyl Sulfoxide BioReagent (DMSO, for molecular biology) and ethidium bromide (EB) were purchased from Sigma-Aldich. 5-diphenyltetrazoliumbromide (MTT) was obtained from Solarbio Science & Technology Co., Ltd (Beijing, China). 0.4% Trypan blue solution was purchased from Amresco (Solon, OH). Micro BCA™ Protein Assay Kit was purchased from Thermo Scientific (Rockford, IL). Luciferase Assay Kit was purchased from Promega (Madison, WI). Water was purified using a Milli-Q instrument (Millipore). Other chemicals used were analytical grade.
Dulbecco’s Modified Eagle’s Medium (DMEM), Fetal Bovine Serum Gold (FBS) and Trypsin-EDTA solution were purchased from PAA. Lyso-Tracker™ Green DND-26 was purchased from Invitrogen. pGL3 luciferase gene siRNA and Allstars Negative Control siRNA (Cat. No. 1027280) were obtained from Qiagen. TAMRA labeled siRNA was purchased from GenePharm. All the materials used for siRNA experiments were processed with DEPC to keep RNase free.

HL60 cells (ATCC, CCL-240, Manassas, VA, USA) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, HyClone, GE Healthcare Life Sciences, SH30228.01, Marlborough, MA, USA) supplemented with 20% fetal bovine serum (FBS, HyClone, GE Healthcare Life Sciences SH30396.03, Marlborough, MA, USA) and 1X Penicillin/Streptomycin (P/S, Caisson Labs, PSL01, Smithfield, UT, USA), as recommended on ATCC website. Jurkat cells (clone E6-1, ATCC, TIB-152, Manassas, VA, USA) were initially cultured in RPMI 1640 with 10% FBS and 1X P/S and gradually changed into IMDM supplemented with 10% FBS and 1X P/S. The cells were all cultured between 1 × 10⁵ cells/mL and 1 × 10⁶ cells/mL. Cell counting was conducted by Invitrogen Countess 3 Automated Cell Counter (Fisher Scientific, A49865, Waltham, MA, USA). Cell viabilities were assessed by Trypan Blue solution (VWR, 45000-717, Radnor, PA, USA). All cells were tested to confirm that they were free of mycoplasma by the Mycoplasma Assay Kit (Agilent Technologies, 302109, Santa Clara, CA, USA) every two weeks.

Cell viability was assessed both with the trypan blue exclusion test and Live/Dead flow cytometry. For the flow cytometry analysis, MSCs in each media formulation were harvested at P5 via digestion with 0.05% trypsin and transferred into a 50-ml conical tube for centrifugation at 200 g for 4 min at room temperature. Following aspiration of excess media, cells were either washed three times with phosphate-buffered saline (PBS) with calcium and magnesium(+/+) and PBS without calcium and magnesium (−/−) or once with PBS (−/−) followed each time by a centrifugation cycle. MSCs were counted using an automated cell counter and stained with 0.4% Trypan blue solution (VWR, Radnor, PA). One million MSCs cultured in FBS or ePL culture media were resuspended in 1 ml PBS and stained with 4 μM ethidium homodimer (Biotium, Fremont, CA) and 2 μM Calcein Blue AM (Thermo Fisher Scientific, Waltman, MA). MSCs stained with either ethidium homodimer or Calcein Blue AM alone were used as control groups. As a negative control, MSCs were harvested, fixed with 4% paraformaldehyde (PFA) for 20 min on ice, washed with PBS, and stained with both ethidium homodimer and Calcein Blue AM. Samples were analyzed by flow cytometry and 50,000 events were collected per sample. Data were analyzed by Flow Jo software (NIH).

mLNs were harvested and placed on a 40 μM filter (Thermo Fisher Scientific) in a 6-well plate with 5 mL complete RPMI. After being dissociated through the filter with the back end of a sterile 1 mL syringe, cells were spun down in 15 mL Falcon tubes at 400g for 5 minutes and then resuspended in 1 mL complete RPMI 1640 before cell counting with trypan blue solution (VWR). Similar cell numbers were plated for flow analysis.