3. If your result falls into any quadrant other than the "High Yield-High Viability" quadrant, refer to Appendix D, Improving Cell Yield and Viability, before proceeding to your next trypsinization.

Maintenance After Subculturing
After 24 hours:

1. Examine the cells microscopically. At least 60% of the cells should have attached to the culture flask.

Some cells will be loosely adherent, but most will have spread out on the culture flask surface. At this stage, most cells will be single or in small colonies.

2. Change the culture medium to remove residual trypsin and non-attached cells.

3. Incubate for an additional 24 hours, and re-examine the culture.

a.At this stage, the vessel should have several mitotic figures indicating that the cells have resumed active growth.

b.If few mitotic figures are observed, contact your Clonetics® Technical Specialist for assistance.

4. Change the medium again 48 hours after the day 1 feeding, and every 48 hours thereafter while examining the culture daily.

5. Feed with volumes as outlined in the table on page 13.

6. Passage again when the cells are 70-90% confluent. (If seeded at the recommended seeding density, this should take 5-9 days.)

Instructions for Proliferating Cells

Cell Preparation: Proliferating Cells
With the proliferating culture of NHDF or NHLF you received, do the following:

1. Examine the culture microscopically for any signs of distress during shipment (i.e., detachment, rounding-up or atypical morphology). Check the relative cell density and estimate "% confluency." The culture should be 30-80% confluent upon receipt. Some cellular detachment is normal. Please call Clonetics® Technical Specialist immediately if cells look severely distressed.

2. Decontaminate the external surface of the cell culture flask by wiping with 70% ethanol or isopropanol.

3. Incubate the sealed flask at 37·C, 5% CO2 for three to four hours to equilibrate temperature.

4. Warm an appropriate amount of growth medium (see table on page 13) to 37·C in a sterile container. Warming the entire bottle can shorten the life of the medium. Never warm growth medium under hot running water or any other uncontrolled temperature source. NEVER MICROWAVE!

5. In a sterile field, carefully open the cell culture flask, remove the medium and replace it with the warmed, fresh growth medium. Aseptically remove any medium inside the neck or cap area because it can facilitate microbial contamination.

6. Loosen the cap, and return the flask to the 37·C humidified incubator with 5% CO2 for at least 24 hours.

Subculturing 
Examine your cultures microscopically every day.

1. Subculture the cells when they reach 70-90% confluency. NHDF and NHLF cultures should have many mitotic figures throughout the flask. Cells should be ready to subculture within 24 to 48 hours, however, shipping conditions such as temperature fluctuations may affect the actual time at which the cells are ready for subculture.

2. Avoid the loss of your culture due to contact inhibition by subculturing cells at no more than 90% confluency. See pages 15-17 for detailed subculturing instructions.

BIBLIOGRAPHY

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.

2) Cytokeratin 18 & 19. Call your Clonetics® Technical Specialist for a reference on this procedure.

3) Wagner, D. D., Olmsted, J.B. and V.J. Marder. (1982) Immunolocalization of Von Willebrand Protein in Weibel-Palade Bodies of Human Endothelial Cells. Journal of Cell Biology., 95:355-360.

4) Grizzle, W.E., and S.S. Polt. (1988) Guidelines to Avoid Personnel Contamination By Infective Agents in Research Laboratories That Use Human Tissues, J. of Tissue Culture Methods, Vol. 11, No. 4.

TECHNICAL SERVICE:

BioWhittaker Inc.
Clonetics® Products
9245 Brown Deer Road
San Diego, CA 92121
(800) 852-5663

INTERNATIONAL TECHNICAL SERVICE:

BioWhittaker Inc.
Clonetics Products
8830 Biggs Ford Rd.
Walkersville, MD 21793-0127
301-898-7025
FAX: 301-845-2924
E-mail: techsup@biowhittaker.com

ORDERS:

BioWhittaker, Inc.
Clonetics® Products
8830 Biggs Ford Road
Walkersville, MD 21793
(800) 344-6618

APPENDIX A
OVERVIEW OF FIBROBLAST MEDIA

500 ml Bottles (except where indicated)

NOTE: All Clonetics® Media can be custom formulated to meet your research needs. Contact your Technical Specialist for more information.

APPENDIX B 
CELL COUNTING USING A HEMACYTOMETER

Background

Proper use of a hemacytometer is critical for obtaining an accurate count of cells and is a procedure used by BioWhittaker to determine the suspension counts for Clonetics® cell strains. A hemacytometer consists of a thic, kened glass slide into which a small chamber has been cut to allow for the introduction of cells to be counted. The floor of the chamber is divided (etched) into nine sections; usually only the four corner sections are used in cell counting (See Figure 1 below). With a coverslip in place, each square of the hemacytometer represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml (and the total number of cells) can be determined.

Procedure

1.Prepare a cell suspension as instructed in step 13 on page 16.

2.Prepare a hemacytometer for use.

a. Carefully clean all surfaces of the hemacytometer and coverslip.

b. Take care to ensure that all surfaces are completely dry using non-linting tissue.

c. Center the coverslip on the hemacytometer.

3.Pipet approximately 9 microliters (this volume will vary slighting with the brand of hemacytometer) of the cell suspension into one of the two counting chambers.

a. Use a clean pipet tip.

b. Be sure that the suspension is thoroughly, but gently, mixed before drawing the samples.

c. Fill the chambers slowly and steadily.

d. Avoid injecting bubbles into the chambers.

e. Do not overfill or underfill the chambers.

4.Count the Cells.

a. Allow the cell suspension to settle for at least 10 seconds.

b. Count all of the cells in each of the four 1 mm3 corner squares labeled A thru D in Figure 1 on the next page.

1) DO count the cells touching the top or left borders.

2) DO NOT count the cells touching the bottom or right borders.

Hemacytometer Reference Figure 1