Basic technique Training and and Practice  Pipetting and transfer of fluid  Observation of cultured cells  Aseptic technique: preparation of mediums and buffers  Feeding a culture  Washing and sterilization of water  Counting cells by haemacytometer  Subculture of continuous cell line– monolayer culture  Construction and analysis of growth curve

Advanced Techniques Detection of mycoplasma Cell line characterization and analysis Primary Culture Cytotoxicity assay

Exercise 1. Aseptic Technique Purpose: Practice in transferring fluid from one bottle to another Exercise 2. Aseptic Technique Purpose: Preparation of Buffers and Culture medium and Procedures : 1.Preparation of 10XPBX 2.Preparation of DMEM+ FBS+ Penicillium/Streptomycin 3.Sterilization of bottles and pipettes and equipments

D-PBSA g/L final concentration KCl KH2PO NaCl Na2HPO4.7H2O Make 500ml/bottle, and autoclave before use

1X DMEM growth medium 1 pack DMEM from sigma add 1000 ml DDW  filter through 0.45um filter  Add 450ml/bottle  Add 50ml FBS, 5 ml P/S

Aseptic Technique Cell Culture Basics

Exercise 3. Cell Line maintenance and cell passage

General procedure for the cell culture laboratory 1. Thawing cells Culture of cell lines( frozen stock)  Thaw  Cell line maintenance

Day 1 frozen culture( -190 o C) 1 ml in frozen medium  37 o C, thaw  Add, 10ml culture medium( DMEM+FBS+p/s)  1200 rpm, 5 min  Remove suspension  resuspend cell pellet with 5 ml growth medium  plating 10 6 cells/ml

Day 2 Culture( cells checked under inverted microscope)  Remove culture medium, Replace with fresh culture medium (3ml)

Day3 subculture  remove culture medium  cells washed with 2ml CMF/PBS  remove PBS  cells, trypsinize with 1x trypsin( 0.025%) ( flush cell by pipetting repeatly)  cells taken into 15ml centrifuge tube  1200 rpm, centrifuge, 10min  trypsin, removed add 5 ml DMEM  cells diluted for plating( 1/4 dilution) in 6 mm cullture dish  37 o C, CO2 incubator

2 to 3 days later cells trypsinize with 1x tyrpsine ( protocols follow the previous page )  cells, resuspend with 5 ml DMEM  cell, 10 ul was taken+ 10ul trypane blue  cell counting

haemacytometer 10 ul cellwas taken+ 10ul trypane blue

(N1+N1)/2 /4 *2 * 10 4 =cells/mlCell number= N1 N2

Counting cells

Cell freezing ( 10 6 cells /ml) confluent cultures  cells trypsinized  cell counting  cell pellet resuspend in frozen medium  -20 o C( 2-4hrs)  -70 o C(2-4hrs)  -196 o C ( DMEM+10%DMSO)

Gradually Lower temperature to -20 o C in ( 2-4hrs)  -70 o C(2-4hrs)  -196 o C Freezing Cells

Cell lines: CHO Chinese Hamster Ovary 3T3 mouse fibroblast CT26 colon carcinoma cell line B16F1 melanoma MG-63 human osteosarcoma skHEPI Hepatoma STO mouse embryonic fibroblast HeLa cervical cancer C2C12 mouse fibroblast

Cell Culture Basics Passaging Cells Thawing Cells Freezing Cells Aseptic Technique Counting cells