细胞实验技术-课堂PPT-第三讲.ppt

Trypan blue staining and cell counting Objectives: What is cell counting? Why do we need to count cells? What is hemocytometer? How to handle hemocytometer? What is trypan blue? What is dilution factor? How to calculate cells? How to perform the procedure? Cell staining and counting Identify a hemocytometer from regular glass slides To determining the number of cells per unit volume. originally designed for performing blood cell counts. Counting grids of hemocytometer By Richard Wheeler (Zephyris) 2007. Hemocytometer grid:red square = 1.0000 mm2, green square = 0.0625 mm2, yellow square = 0.040 mm2, blue square = 0.0025 mm2,  at a depth of 0.1 mm 1 2 3 4 5 6 7 8 9 25格×16格的血球计数板计算公式： 细胞数/ml=4大格内细胞个数/4×10000×稀释倍数 细胞计数 1、将血球计数板及盖片用擦试干净，并将盖片盖在计数板上。 2、将细胞悬液吸出少许，滴加在盖片边缘，使悬液充满盖片和计数板之间。 3、静置2-3分钟。 4、镜下观察，计算计数板四大格细胞总数，压线细胞只计左侧和上方的。然后按下式计算： 细胞数/ml ＝ 4大格细胞总数 ×10000 4 注意：镜下偶见由两个以上细胞组成的细胞团，应按单个细胞计算，若细胞团占10%以上，说明分散不好，需重新制备细胞悬液。 Trypan blue live cell membrane is impermeable to trypan blue, but dead cell membrane absorbs trypan blue. Hence trypan blue is used in cell viability test. Dilution of cells…. In order to count cells… Detach cells from the flask. Pipette and mix well to remove clumped cells. Collect small amount [ 100 μL to 500 μL] in eppi tube. Add trypan blue into the cells in eppitube. Depending on the amount of trypan blue calculate the dilution factor. Dilution factor = Final volume / initial volume. Ex. 100 μL of cells and 100 μL of trypan blue DF= 200 μL / 100 μL = 2 To prepare the counting chamber The mirror-like polished surface is carefully cleaned with lens paper. The cover slip is also cleaned. The cover slip is placed over the counting surface prior to putting on the cell suspension. counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power. One entire grid on standard hemocytometer can be seen at 40x Clean the cover slip