BY-细胞的培养与转化.docVIP

BY-2 Cells: Culture and Transformation for Live Cell Imaging BASIC PROTOCOL 1 ESTABLISHING A BY-2 SUSPENSION CULTURE BY-2悬浮培养的建立 This protocol describes the method to establish a BY-2 cell suspension from a BY-2 or another tobacco callus. Subculturing the cells is technically termed passaging (also see Basic Protocol 2). It is advisable to work in duplicate or even triplicate when establishing a suspension culture. This allows initiation of fresh suspensions of the same transformed cell line should any contamination occur. 这个实验步骤描述了通过BY-2或者其他的烟草愈伤组织建立BY-2细胞悬液的方法。传代培养细胞是一个传代的专有名词。当建立悬浮体系的时候最好是一式两份或一式三份的进行。这样就使得一旦一个转化的细胞株的污染发生的时候，又可以开始新的细胞悬浮。 Materials Calli of wild-type and/or transformed BY-2 cells (available from various laboratories) grown on solid BY-2 medium (see recipe) in petri dish 50-ml conical flasks containing 20 ml liquid BY-2 medium (see recipe), covered with aluminum foil and autoclaved Suitable filter-sterilized antibiotics, for culturing transformed BY-2 cells only Razor blades, sterile Packet of aluminum foil squares for covering flasks, sterile Shaking incubator, 25?C 1. Cut a 2-cm2 piece of wild-type or transformed BY-2 callus with a sterile razor blade directly on the petri dish on which the calli are growing. 2. Loosen aluminium foil from top of a sterile 50-ml conical flask containing 20 ml liquid BY-2 medium. Lightly flame neck of flask. Add suitable filter-sterilized antibiotics if needed. Open a sterile packet of aluminum foil squares so that they are readily accessible. 材料 转化过的或野生型的BY-2细胞的愈伤组织（不同的实验室都有）生长于培养皿的BY-2固体培养基上（见配方） 装有20ml液体BY-2培养基（见配方）的三角瓶，用铝膜纸封口高温灭菌。 过滤灭菌-转化BY-2细胞相关的抗生素 无菌的保险刀片 一捆方形的铝膜纸用来包烧瓶的，灭菌 25度摇床 直接在培养基上用灭菌的刀片切2平方厘米的野生型或者是转化的BY-2愈伤组织，并让其在培养基上生长。 松开灭菌的装有液体培养基的三角瓶的铝膜封口，用火烧瓶口，加入合适的抗生素。打开一个正方形的铝膜纸，先做好准备。 This will aid the speed at which the items in the hood can be handled and hence will reduce the possibility of contamination. The flasks should be open for as short a time as possible to avoid contamination. Avoid passing