Calcein am pi double stain kit

Poly (lactic-co-glycolic acid) (PLGA), IR780 iodide, and polyvinyl alcohol (PVA) were all obtained from Sigma-Aldrich (USA). Doxorubicin hydrochloride (Dox) was purchased from Solarbio Co., Ltd. (China). Both DiI stain kit and BCA protein assay kit were purchased from Beyotime Inst. Biotech (China). The platelet protein extraction kit was purchased from BesBio (China). The Calcein-AM/PI Double Stain Kit was obtained from Yeasen (China). The Cell Counting Kit-8 (CCK-8) was obtained from 7sea Biotech (China). All experiments were approved by the Ethics Committee of the Second Xiangya Hospital, Central South University, China.

iRGD and FFVLK-(PEG8)₃ (MW = 1923.2 Da) were synthesized by Shanghai Dechi Biosciences Co., Ltd. (Shanghai, China). Ce6 was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Bilirubin was gained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). d-SN38 was supplied by Prof. Jun Cao (Sichuan University, China). 4′,6-Diamidino-2-phenylindole (DAPI) and Lysotracker green was purchased from Life Technologies (Grand Island, USA). FITC Annexin V Apoptosis Detection Kit I was obtained from BD Pharmingen™ (Franklin Lakes, NJ, USA). Calcein-AM/PI Double Stain Kit was supplied by Yeasen (Shanghai, China). 24-well hanging cell culture inserts (8.0 μm, PET) were obtained from Corning Inc. (Kennebunk, ME, USA). All other reagents and solvents were purchased from commercial resources and used without further purification unless otherwise noted. Mouse mammary breast tumor cell line 4T1 was obtained from Chinese Academy of Sciences Cells Bank (Shanghai, China). 4T1 cells were incubated in RPMI-1640 cell culture medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL streptomycin, and 100 U/mL penicillin at 37 °C in a humidified 5% CO₂ atmosphere.

Cell survival on the surfaces of scaffolds with different growth factors was assessed by live/dead assay (Calcein-AM/PI Double Stain Kit, YEASEN, Shanghai, China). Briefly, after exposure to TGF-β1 (10 ng/mL), IGF-1 (10 ng/mL), or GDF-7 (100 ng/mL) (PeproTech, NJ, United States) for 12 d, the BMSC-collagen sponge constructs were harvested and incubated with 2 μmol/L calcein AM and 4.5 μmol/L propidium iodide (PI) for 30 min in the dark at 37 °C. After rinsing three times with PBS, the fluorescence was visualized using a fluorescence microscope (Olympus, Japan).

The effect of the PSP composites on cell proliferation was evaluated with a CCK-8 assay (DOJINDO, Japan) by following the manufacturer's protocol. Briefly, the extract solution was prepared by soaking the materials in an USCs medium (area: volume = 1 : 6) at 37 °C for 24 h. Meanwhile, the USCs were seeded on a 96-well plate at a density of 5 × 10⁴ cells/well and cultured with the medium. After 24 h of culture, the medium was replaced with extract solution. Their viability was tested using a CCK-8 assay kit on days 1, 3, 5, and 7. Bovine pericardium (Synovis Surgical Innovations Inc, USA), a commercially made cardiac patch, was used as the control. Viability of the cells on the PSP composites was evaluated with a Calcein-AM/PI Double Stain Kit (Yeasen, China). In brief, the P3 USCs were seeded onto the PSP composites at a density of 2 × 10⁴ USCs/cm². Cell/scaffold constructs were incubated in sterile PBS containing 2 mM Calcein-AM and 4 mM ethidium homodimer-1 for 20 min at 37 °C in the darkness after 1, 3 and 5 days of culture. The samples were then observed under a confocal microscope (Nikon A1RMP+, Nikon Instruments Inc., Tokyo, Japan).

48 h after the 3D culturing of human dermal fibroblasts in the syringe tubes, external pressure force was added by sealing the syringe cap and compressing the syringe piston with a self‐assembled device shown in Figure 1. The 3D‐cultured human fibroblasts were then pressurised under 1.5 atm for 4, 8, and 20 h respectively. For the control tubes, the syringe caps were sealed without adding extra force. Calcein‐AM/PI Double Stain Kit (Yeasen, China Cat#40747ES76) was used to stain the cells before and after pressure intervention, to discriminate live/dead cells, therefore, examine the viability of the fibroblasts during this experiment. Immediately after completing the pressure intervention, the culture medium was discarded, and the gel‐cell mixture was collected and kept in either TRIzol (Tiangen, China Cat#DP424) reagent or RIPA Lysis Buffer (Servicebio, China Cat# G2033) supplemented with proteinase inhibitors for further RNA and protein extractions respectively.