FibroblastCellSystems6

APPENDIX E 
GROWTH AREA OF COMMON PLASTICWARE

APPENDIX F 
Seeding Into Multi-Well Plates

Overview

A culture flask of normal human cells is harvested by trypsinization and subsequent trypsin inhibitor treatment. The cells are centrifuged, resuspended in growth medium and counted. The desired number of cells is then added to wells of sterile Multi-well tissue culture plates. The plates are incubated in a 37oC, 5% CO2 humidified incubator for one to three days to allow for cell adherence and growth. Seeding densities will vary somewhat with your experimental requirements. We recommend a density for Dermal Fibroblasts and Lung Fibroblasts of 10,000 cells/cm2 for all multiwell plates.

Required Materials:

1. T-25 flask of proliferating normal human cells between 70% and 90% confluence.

2. Flat-bottom, Multi-well tissue culture plates

3. 37oC humidified incubator with 5% CO2 /95% air

4. Laminar flow hood or other sterile environment

5. Adjustable multichannel pipetter (8- or 12-channel) or repeating pipetter

6. Sterile reservoir(s) for use with multichannel pipetter

Procedure

1. Follow the steps on pages 13-15 for subculture preparation and subculturing. Then follow steps 2-4 below.

2. Since the cells/ml calculation computed on p. 20 is "per ml", one must increase the cell concentration by 4 times before seeding 96 well plates (to accommodate the 1:4 dilution when adding 250 ul of suspended cells per well). When making the cell suspension, adjust the cell concentration with growth medium.

3. Transfer the diluted cell suspension to a sterile reservoir. Using a multichannel (8- or 12-channel) pipetter equipped with sterile pipette tips, add 250 ml of the diluted cell suspension to each well of the labeled 96- well, flat bottom, tissue culture plate(s). RESUSPEND THE CELL SUSPENSION OFTEN DURING THE SEEDING PROCEDURE TO ENSURE A UNIFORM NUMBER AND DISTRIBUTION OF CELLS INTO EACH WELL BY PIPETTING UP AND DOWN A FEW TIMES BETWEEN EVERY OTHER DISPENSING.

4. Cover and incubate the plates for 1 to 3 days at 37oC/5% CO2. (Incubation periods exceeding 3 days are generally not recommended because of evaporation of medium from the edge wells of the plate.)

NOTE: Before using the Multi-well plate culture in a bioassay, examine them microscopically for the presence of mitotic figures as a confirmation that the cells have resumed active growth. (Does not apply for all end-user assays.)

Fax Order Form

(Please print or type)

Name of Principal Investigator:

Date Required:

Phone Number to Confirm Ship Date:

(1)Polymerase Chain Reaction (PCR) technology is covered by U.S. Patents 4,683,195, 4,683,202, and 4,965,188 owned by Hoffman La-Roche, Inc.