本产品包含各种常用的蛋白酶,能特异性地降解胞外胶原蛋白,使胶原蛋白和纤维粘连蛋白的网状结构解离,而不损坏细胞表面结构的完整性。使用本产品进行类器官消化可以使类器官细胞间的桥连结构松动,使类器官变得松散,经过机械吹打后制成细胞群团悬液。制备的细胞悬液可用于类器官扩大培养、流式细胞仪进行细胞生物学 (如凋亡、增殖、细胞周期等)研究。
适用范围:各种类型的类器官。
使用说明
1. 去除类器官培养板中的培养基,加入1 mL预冷PBS吹打基质胶,反复3-5次;
2. 将悬液转移至15 mL离心管中,1500 rpm常温离心5 min;
3. 小心吸去上清和基质胶层,可重复加入预冷PBS吹打,离心,最大程度去除基质胶。
4. 弃上清,加入5-10 mL解离试剂,封口后放入恒温振荡培养箱中消化,设置温度37℃、转速200~300 rpm (根据类器官大小设置转速),注意镜下检查类器官消化情况,类器官消化成多细胞团块为宜;
5. 取出消化好的样本,1500 rpm常温离心3 min。
6. 弃上清后,加入6 mL含血清培养基进行终止消化,1500 rpm室温离心3 min。
7. 弃上清后加入稀释液待用。
注意事项
1. 使用产品时应注意无菌操作,避免污染。
2. 为保持本产品的最佳使用效果,一般在37℃条件下使用,根据具体实验情况也可在2-8℃条件下使用。
3. 请于保质期内使用本产品,超出保质期后,将无法达到理想的使用效果。
4. 本产品仅用于科研用途,不能用于体外诊断。
This product contains a variety of commonly used proteases, which can specifically degrade extracellular collagen and dissociate the reticular structure of collagen and fibronectin without damaging the integrity of cell surface structures. Organoid digestion with this product loosens intercellular bridging connections within organoids and disperses organoid masses. A cell cluster suspension can be obtained after mechanical pipetting. The prepared cell suspension is applicable for organoid expansion culture and flow cytometry-based cell biology researches, including apoptosis, proliferation, cell cycle analysis and other assays.
Application Scope
All types of organoids.
Instructions for Use
1. Remove culture medium from organoid culture plates, add 1 mL pre-cooled PBS to pipette and wash Matrigel repeatedly for 3–5 times.
2. Transfer the suspension into a 15 mL centrifuge tube, and centrifuge at 1500 rpm at room temperature for 5 minutes.
3. Carefully aspirate the supernatant and Matrigel layer. Repeat pre-cooled PBS rinsing, pipetting and centrifugation to remove Matrigel as completely as possible.
4. Discard the supernatant, add 5–10 mL dissociation reagent, seal the tube and place in a constant-temperature shaking incubator for digestion. Set parameters at 37℃, 200–300 rpm (adjust speed according to organoid size). Monitor digestion status under microscope; digestion is optimal when organoids are dissociated into multicellular aggregates.
5. Take out digested samples and centrifuge at 1500 rpm at room temperature for 3 minutes.
6. Discard the supernatant, add 6 mL serum-containing medium to terminate digestion, and centrifuge at 1500 rpm at room temperature for 3 minutes.
7. Discard the supernatant and add diluent for subsequent use.
Precautions
1. Strict aseptic operation is required during use to avoid contamination.
2. To maintain optimal performance, the product is generally used at 37℃. It can also be applied at 2–8℃ according to actual experimental conditions.
3. Use the product within the shelf life. Desired experimental res