CYTOSPIN PREPARATION (Appendix XX)

I. Introduction

A cytospin preparation is a concentration of cells taken directly from specimens or from scraped cell cultures.

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II. Reagents and Materials

Virus-specific or pooled antibody

Phosphate buffered saline (PBS)

Cold acetone (4^oC)

Distilled water

Mounting fluid

Non-immune antibody

vortex

sterile pipettes

cytospin and accessories

humidified chamber

coplin jars

fluorescence microscope

 

III. Procedure

1. Shell Vial

i. Remove all except 1 ml maintenance media from shell vial using a sterile pipette.

i. Scrape cells from top of coverslip using a sterile pipette. Break up cell clumps by pipetting the cells up and down several times.

ii. Pipette 200 ul (4 drops) of scraped cells into funnel for each well.

iii. Cytospin at 2000 rpm (700g) for 5 minutes.

iv. Remove slide and air dry.

v. Fix in cold acetone for 10 minuets in a coplin jar. Remove slide and air dry.

Proceed to staining. Refer to Appendix IV for Indirect fluorescent antibody staining techniques or Appendix V for Direct fluorescent antibody staining techniques.

or

Refer to Appendices IV and V for immunofluorescent staining techniques for shell vials.

 

1. Tube culture (or Shell Vials for CPE)

i. Remove all except 1 ml maintenance media from the culture tube using a sterile pipette.

ii. Scrape cells from side of tube using a sterile pipette. Break up cell clumps by pipetting the cells up and down several times.

iii. Pipette 200 ul (4 drops) of scraped cells into funnel for each well.

iv. Cytospin at 2000 rpm (700 x g) for 5 minutes.

v. Remove slide and air dry.

vi. Fix in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.

vii. Proceed to staining. Refer to Appendix IV for Indirect fluorescent antibody stains or Appendix V for Direct fluorescent antibody stains.

 

or

Refer to Appendices IV and V for immunofluorescent staining techniques for shell vials.

 

3. Direct from specimen

IV. Reference

Thermo Shandon, cytospin. Manufacturer's manual. Refer to Appendix VI for procedure.

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