Proliferation of Human Mononuclear Cells with CFSE

Transcription

1 UCANU 0005 Version 01, November 2010 Page 1 of 7 CMCI (Center for molecular and Cellular Intervention) University Medical Center Utrecht Written By Name Function Date Signature Mark Klein Lab manager Conformation Name Function Date Signature Prof.dr. A.B.J. Prakken CoChair CMCI Changes from last version Date of version Paragraphs Date of version Paragraphs

2 UCANU 0005 Version 01, November 2010 Page 2 of 7 Content 1. Subject Application Definitions and Abbreviations Principle Safety precautions Reagents Chemicals Basic Culture Medium Wash Medium % FCS Equipment and Accessories tools Equipment Accessories Samples Sample Collection Sample Processing Procedure Documentation Accuracies and Precision Accompanied Forms/ Attachments/ Document Remarks Literature... 6

3 UCANU 0005 Version 01, November 2010 Page 3 of 7 1. Subject This Standard Operation Procedure (SOP) describes a method how to use the intracellular fluorescent dye CFSE to monitor lymphocyte proliferation 2. Application The stable incorporation of the intracellular fluorescent dye 5(and 6)carboxylfluorescein diacetate succinimidyl ester (CFSE) into lymphocytes is a powerfool tool to monitor lymphocyte migration in vivo and to quantitatively analyze cell divisions both in vivo and in vitro. 3. Definitions and Abbreviations CFSE = 5(and 6)carboxylfluorescein diacetate succinimidyl ester CFDA,SE = Carboxyfluorescein diacetate, sucinimidyl ester 4. Principle In order to fully understand a functioning immune system, techniques are required that can simultaneously follow lymphocyte proliferation. [ 3 H]thymidine has been widely used to follow proliferation, both in vivo and in vitro. However, this approach suffers from the disadvantage that it is difficult to assess the subpopulation of lymphocytes that is proliferating. Furthermore, the procedure only measures those cells that are in the S phase at the time of [ 3 H]thymidine addition. CFSE is a membranepermeant dye that can very stably label cells by covalently coupling to intracellular molecules. A major advantage of CFSE is that it can be used to monitor lymphocyte proliferation, due to the progressive halving of CFSE fluorescence in cells following cell division. Figure 1: Mechanism of cellular labeling by CFDA,SE. CDFA,SE is annonpolar molecule that spontaneously penetrates cell membranes and is converted to anionic CFSE by intracellular esterases. Aminereactive coupling of CFSE to proteins results in table longterm intracellular retention Carboxyfluorescein diacetate, sucinimidyl ester (CFDA,SE) spontaneously and irreversibly couples to cellular proteins by reaction with lysine sidechains and other available amines (see figure 1). After an initial period of equilibrium (~24 hours), the fluorescence of resting cells labeled with CFDA,SE is stable over periods of months and can be analyzed by flow cytometry using excitation at 488 nm and the Fl1 detection channel. When cells divide, CFDA,SE labeling is distributed equally between the daughter cells, which are therefore half as fluorescent as their parents. Consequently, each successive generation in a population of proliferating cells is marked by specific twofold decrements in cellular fluorescence intensity that are readily discriminated by flow cytometry. (See Attachment 1) Although current applications of CFSE laeling are centered on lymphocyte proliferation, the

4 UCANU 0005 Version 01, November 2010 Page 4 of 7 technique is also applicable to other cell types, including fibroblasts, NK cells and even bacteria 5. Safety precautions Treed every sample containing human material such as AB serum and the lymphocyte samples as infectious material. Wear disposable gloves. NOTE: Be sure to wear a fullface shield during the thawing procedure because cryovials containing liquid nitrogen have been known to explode SumaTab contains sodiumdichloroisocynate Trypan blue (0.4%) DryIce Ethanol CFDA,SE (CFSE) Xn, Harmful T, Toxic Xn, Harmful F, Flammable Xn, Potentially harmful 6. Reagents 6.1 Chemicals Reagents Formula Supplier order number Store at ( C) RPMI PBS Pharmacy (WKZ) RT Lglutamine PenicillinStreptomycin Foetal Bovine Serum Trypan Blue (0.4%) C 34 H 24 N 6 O 14 S 4 Na RT Ethanol CH 3 OH Pharmacy (WKZ) RT CFSE C 29 H 19 NO 11 C C 6.1 Basic Culture Medium RPMI 1640 supplemented with 1% v/v P/S (= 5ml) and 1% v/v glu (=5ml) Store at 4 C up to 1 month

5 UCANU 0005 Version 01, November 2010 Page 5 of Wash Medium RPMI 1640 supplemented with 1 v/v% P/S (= 5ml), 1 v/v% glu (=5ml) and 2% v/v FBS (=10 ml) FBS should be heat inactivated (1 hr at 56 C) and filtered through a 0.20 µm sterile filter using a 10 ml sterile syringe before usage. Store at 4 C up to 1 month % FCS Basic culture medium supplemented with 10 v/v% FBS. FBS should be heat inactivated (1 hr at 56 C) and filtered through a 0.20 µm sterile filter using a 10 ml sterile syringe before usage. Store at 4 C up to 1 month. 7. Equipment and Accessories tools 7.1 Equipment Centiguge Hettich Rotanta 46 (UMC# ) Reichert brightline hemacytometer (Hausser Scientific Company, Horsham PA, USA) Easypet pipet (Eppendorf, Germany, ) Pipets µl (Gilson, The Hague, The Netherlands) Multistep pipet (Eppendorf, Germany) 7.2 Accessories 50 ml sterile polypropylene conical tubes (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) Sterile 10 ml syringe (Becton Dickinson, Erembodegem, Belgium) 2 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 5 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 10 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) 25 ml sterile disposable pipet (Falcon/ Becton Dickinson, Erembodegem, Belgium, ) Disposable gloves (Kimberly Clark, Zaventem, Belgium) 8. Samples 8.1 Sample Collection The amount of cells frozen and other data concerning a sample can be found in the cell storage 8.2 Sample Processing All handlings of the sample should be done in a biohazard safety cabinet 9. Procedure 1. Wash cells 3 times in PBS 2. Resuspend freshly isolated and counted lymphocytes in PBS, final concentration from 10x10 6 cells/ml to 30x10 6 cells/ml 3. Dilute stocksolution CFSE (10mM) 100x in PBS to give a 100 µm solution CFSE (5 µl 10 mm CFSE µl PBS) 4. Dilute the predilution of CFSE (100 µm) 50x in the cell suspension (for a final concentration

6 UCANU 0005 Version 01, November 2010 Page 6 of 7 of 2 µm) 5. Mix the solution rapidly and incubate 10 min at 37 C (incubator), set centrifuge temperature to 4 C. 6. Add 10 volumes of icecold filtered 100% FBS 7. Centrifuge the cells 10 min at 1600 rpm (4 C) 8. Repeat step #7 three times with 10 volumes 10% FCS medium 9. Resuspend the cells in culture medium and continue with the T cell proliferation assay 10. Documentation Document each sample in used at your notebook. Remove the used vial with cells from the cellstorage 11. Accuracies and Precision Accuracy is hard to describe. Cell counts should be in a range of 75% of the original frozen cell numbers. Furthermore if the number of dead cells are higher then 20% it is desirable to run the sample over a ficoll grandient again. 12. Accompanied Forms/ Attachments/ Document Attachment 1 : CFSE labeling lymphocytes 13. Remarks Cryovials are submerged in liquid nitrogen. So when a vial is note properly closed it will fill partly with nitrogen. When the vials a placed directly in warm water without letting the nitrogen to evaporate by removing the lid of the vial, the nitrogen will expand and the vial will explode! 14. Literature N/A *** END ***

7 UCANU 0005 Version 01, November 2010 Page 7 of 7 Attachment 1: CFSE labeling lymphocytes Resting B lymphocytes labeled wih CFDA,SE were stimulated under conditions designed to resproduce T celldependent activation. 2, 3 and 4 days after stimulation, cells were harvested and analyzed by flow cytometry. Peaks from right to left in the cellular fluorescence histogram represent successive generations. The observation of multiple peaks on days 3 and 4 is indicative of highly asynchronous division.