UNIVERSITY OF LEICESTER CANCER STUDIES & MOLECULAR MEDICINE STANDARD OPERATING PROCEDURE

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1 Page 1 of 4 UNIVERSITY OF LEICESTER CANCER STUDIES & MOLECULAR MEDICINE STANDARD OPERATING PROCEDURE SOP-548 VERSION-03 TITLE: Preparation of cytospins Written by: Angie Gillies Reviewed by Malcolm Rae Changes introduced: 1.Change to CSMM format. Implementation Date: Review Date: PURPOSE To provide a method for the preparation of cytospins from cell suspensions. 2. SPECIAL NOTES 2.1 Full details of the carrying out of this technique must be recorded in the relevant laboratory notebook in accordance with SOP All cultured cells must be regarded as potentially pathogenic and handled in accordance with the laboratory safety manual. 3. CROSS REFERENCES 3.1 Preparation of chrome alum gelatin coated slides SOP Preparation of silane treated slides SOP Cell counts and viability using a haemocytometer SOP Basic cell culture CPA EQUIPMENT AND MATERIALS 4.1 Centrifuge MSE, Super Minor 4.2 Balanced salt solution (BBS) e.g. RPM 137b Dulbecco s phosphate buffered saline (DPBS) Gibco Hanks balanced salt solution (HBSS) Gibco Cytospin fluid Shandon For immunocytochemistry - Chrome alum gelatin coated slides SOP 305 For in-situ hybridisation - Silane coated slides SOP Cytospin funnels and clips 4.6 Cytospin machine Shandon Cytospin % Formal saline RPM 78

2 Page 2 of Acetone Fisher A/0560/ Disinfectant : e.g. Presept disinfectant tablets Surgicos PR 25 Dissolve 1 tablet in 200ml tap water in a large beaker 5. PROCEDURE There are differences in preparative technique dependent upon the eventual use planned for the prepared slides. Slides intended for in situ hybridisation will be mounted on silane coated slides and fixed in formal saline. Slides intended for immunocytochemistry will be fixed in acetone, 70% ethanol or formal saline and can be prepared on subbed slides. A second variation occurs when cells are prepared for in-situ work. For optimum staining the cells will be fixed in formal saline before the slides are prepared. For optimum recovery of cells fixation will occur after slide preparation. In any given project it is likely that both preparative techniques will be used. Cytospin preparation for immunocytochemistry 5.A.1 Count the cell population using a haemocytometer following the instructions in SOP A.2 The culture medium must first be removed and the cells washed as follows: 5.A.2.1 Centrifuge at 800rpm for 5 minutes. 5.A.2.2 Decant supernatant, and re-suspend cells in balanced salt solution. 5.A.3 Centrifuge again at 800g for 5 minutes, and decant supernatant. 5.A.4 Re-suspend the cells in cytospin fluid to produce a cell density of 2x10 6 cells/ml. 5.A.5 Label appropriately treated slides, and place in a cytoclip along with cytospin 5.A.6 Add 500 l of the appropriate cell concentrate to the cytospin funnel and spin 5.A.7 Remove the cytospin preparations from the centrifuge. Allow to air dry and store in a covered tray at room temperature for storage, or fix immediately for 15 minutes, then perform immunocytochemistry. 5.A.8 Discard the cytospin papers into an autoclave bag. Immerse clips and funnels then rinse and air dry.

3 Page 3 of 4 Cytospin preparation for in-situ hybridisation I) With Prefixation 5.B.1 Count the cell population using a haemocytometer following the instructions in SOP B.2 Centrifuge cells at 800rpm for 5 minutes. 5.B.3 Decant supernatant, and re-suspend cells in 10% formal saline to a concentration of 2x10 6 cells/ml. 5.B.4 Allow to fix at room temperature for 15 minutes. 5.B.5 Label appropriately treated slides, and place in a cytoclip along with cytospin 5.B.6 Add 500 l of the appropriate cell concentrate to the cytospin funnel and spin 5.B.7 Remove the cytospin preparations from the centrifuge. Place in 95%IMS for 2 minutes, then in 99%IMS for 2minutes. Then allow to dry air dry and store at room temperature. 5.B.8 Discard the cytospin papers into an autoclave bag. Immerse clips and funnels then rinse, and air dry. I) With Post-fixation 5.C.1 Count the cell population using a haemocytometer following the instructions in SOP C.2 Adjust cell density to approximately 2x10 6 cells/ml. Either centrifuge cells at 800rpm for 5 minutes and decant part of the supernatant or dilute cell suspension with HBSS. 5.C.3 Label appropriately treated slides, and place in a cytoclip along with cytospin 5.C.4 Add 500 l of the appropriate cell concentrate to the cytospin funnel and spin 5.C.5 Quickly remove the cytospin preparations from the centrifuge. Place in 10% formal saline for 15 minutes. 5.C.6 Transfer slides to 95%IMS for 2minutes. Then into 99%IMS for a further 2 minutes. Air dry the slides and store at room temperature.

4 Page 4 of 4 5.C.8 Discard the cytospin papers into an autoclave bag. Immerse clips and funnels then rinse, and air dry.

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