For EdU staining, EdU was added to the culture medium for 4 h in order to incorporate into replicating cells' DNA. Cells were washed and then fixed with 4% paraformaldehyde for 15 min. 0.2% Triton X-100 was used to permeabilize the nuclear membrane. Ultimately, cells were stained by Cell-LightTM Apollo488 Stain Kit (RiBoBio, China) according to the manufacturer's instructions. Cells were detected with a fluorescence microscope (Leica, Germany). For cell number counting, at least 200 cells or 10 images were quantified in each well to get accurate numbers for each group.
Cell light apollo488 stain kit
Manufactured by RiboBio
Sourced in China
The Cell-LightTM Apollo488 Stain Kit is a laboratory product designed for staining and visualizing cellular structures. It provides a reagent for labeling specific cellular components, allowing for their detection and analysis using fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Leica (1 mentions)
Modfit lt 3, by Verity Software House (1 mentions)
Ti u fluorescent microscope, by Nikon (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Anti cd8 apc cy7 clone 53 6.7, by BD (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
4 protocols using cell light apollo488 stain kit
1
EdU Incorporation and Fluorescence Staining
2
Quantifying Cell Proliferation and Cell Cycle
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EdU staining assay (Cell-LightTM Apollo 488 Stain Kit, RiboBio) was used to detect cell proliferation following the manufacturer’s protocol. Briefly, transfected cells were seeded in 96-well plates and incubated with DMEM medium containing EdU for 2 h. Following cell fixation (4% formaldehyde) and cell membrane permeation (0.5% Triton X-100), the cells were stained with Apollo dye solution and Hoechst33342. The images were photographed with a Nikon Ti-U fluorescent microscope (Tokyo, Japan). The EdU-positive cells were counted using Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA, USA).
For the cell cycle assay, transfected cells were collected and fixed with 75% pre-chilled ethanol for 24 h at 4 °C. Then the cells were stained with RNase A/PI (propidium iodide) mixture and incubated in the dark at 37 °C for 30 min. The cells were detected by a CytoFLEX flow cytometer (Beckman Coulter, CA, USA). The cell cycle was analyzed with Modfit LT32 software (Verity Software House, Topsham, ME).
For the cell cycle assay, transfected cells were collected and fixed with 75% pre-chilled ethanol for 24 h at 4 °C. Then the cells were stained with RNase A/PI (propidium iodide) mixture and incubated in the dark at 37 °C for 30 min. The cells were detected by a CytoFLEX flow cytometer (Beckman Coulter, CA, USA). The cell cycle was analyzed with Modfit LT32 software (Verity Software House, Topsham, ME).
3
Tracking Proliferation and Apoptosis in Transplanted Mice
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Recipient mice were pulsed intraperitoneally with 0.1 mg of 5-ethynyl-2′-deoxyuridine (EDU, RIBOBIO) in PBS 10 days after transplantation. 24 h later, spleen and LN cells were isolated and single-cell suspensions were prepared for EDU detection using Cell-Light™ Apollo®488 Stain Kit (RIBOBIO) according to the manufacturer’s instructions. Then, cells were stained for cell surface markers with anti-CD8-APC-Cy7 (Clone 53-6.7), CD44-V450 (Clone IM7), and CD62L-APC Abs (Clone MEL-14) (all from BD Biosciences). To detect cell apoptosis, cells were stained for the same surface markers and then Annexin V-FITC according to the protocol of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen). Cells finally were analyzed via a flow cytometer (FACSCalibur).
4
Histological Analysis of Bone Samples
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Tissues were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (PH 7.8) for decalcification. Then specimens were embedded in paraffin and sectioned at 6 μm. In brief, the samples were immersed in xylene, ethanol, 70% ethanol, and water for dewaxing. For Safranin O staining, the dewaxing samples were immersed in 0.05% Fast green for 3 min, 1% acetic acid for 10 s, 0.5% Safranin O for 5 min, 95% ethanol for 10 s, and water for 5 min. For Von-kossa staining, the dewaxing samples were immersed in silver nitrate for 5 min, water for 5 min for three times, 5% sodium thiosulfate for 5 min, water for 5 min for three times, and Nuclear fast red for 5 min, water for 5 min for three times. Slides were mounted with neutral balsam. Images were taken using a microscope (Olympus BX51, Tokyo, Japan). For TRAP staining, the slides were immersed in the staining buffer at 37°C for 20 min. The staining buffer was a mixture of A buffer (125 ul 10 mg/ml Naphthol AS-MX in 12.5 mL 0.1 M acetate buffer), B buffer (125 µl 15 mg/ml Fast Red LB in 12.5 mL 0.1 M acetate buffer) and 1.5 mL 1 M Sodium Tartrate buffer. For the Edu staining, 0.05 mg/g Edu was injected into the mice at P0. After 24 hours, the mice were sacrificed and the femurs were fixed, decalcified, and embedded in paraffin. The slides were stained using the Cell-Light Apollo 488 stain kit (RIBOBIO, C10371-3).
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