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 0.2 2.68

KH2PO4 0.2 1.47

NaCl 8 136.9

Na2HPO4.7H2O 2.2 8.06

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

http://www.youtube.com/watch?v=4mKhULnxqcw&feature=related

Aseptic Technique

http://www.youtube.com/watch?v=_fjZ-MHV22w

Cell Culture Basics

Exercise 3. Cell Line maintenance and cell passage

General procedure for the cell culture laboratory1. Thawing cells

Culture of cell lines( frozen stock) Thaw Cell line maintenance

Day 1 frozen culture( -190oC) 1 ml in frozen medium 37oC, thaw Add, 10ml culture medium( DMEM+FBS+p/s)

1200 rpm, 5 min

Remove suspension resuspend cell pellet with 5 ml growth medium plating 106 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 37oC, 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 * 104=cells/mlCell number=

N1

N2

http://www.aqualex.org/html/onedin/fish_haematology_no/y4g5.jpg

http://www.youtube.com/watch?v=-cGPS5ryg14&feature=related

Counting cells

Cell freezing ( 106 cells /ml)

confluent cultures cells trypsinized cell counting cell pellet resuspend in frozen medium -20oC( 2-4hrs) -70oC(2-4hrs) -196oC ( DMEM+10%DMSO)

Gradually Lower temperature to -20oC in ( 2-4hrs)

-70oC(2-4hrs)

-196oC

http://www.youtube.com/watch?v=ZtElKq85OuM&feature=related

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

http://www.youtube.com/watch?v=_fjZ-MHV22w

Cell Culture Basics

http://www.youtube.com/watch?v=gaG15lM1t5A&feature=related

Passaging Cells

http://www.youtube.com/watch?v=CCWrLUA6Qbg&feature=related

Thawing Cells

http://www.youtube.com/watch?v=ZtElKq85OuM&feature=related

Freezing Cells

http://www.youtube.com/watch?v=4mKhULnxqcw&feature=related

Aseptic Technique

http://www.youtube.com/watch?v=-cGPS5ryg14&feature=related

Counting cells