Immunofluorescent staining and flow cytometric analysis of cells

Transcription

1 UCAN-U 0003 Version 1 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 Co-Chair CMCI Changes from last version Date of version Paragraphs Date of version Paragraphs

2 UCAN-U 0003 Version 1 November 2010 Page 2 of 7 Content 1. Subject Application Area s Application Normal Values Minimum and maximum values Protocols which follow up this assay Definitions, Terms and Abbreviations Principle Safety precautions Reagents Chemicals Facs Buffer (PBS with 2% FCS and 0.1% Sodium Azide) GolgiStop (Pre-dilution) Culture Medium (RPMI with P/S/G and 2.0% FCS) Fixation buffer (PBS + 1.0% Paraformaldehyde) Equipment and Accessories tools Equipment Accessories Samples Sample Collection Sample Processing Procedure Documentation Quality Control Internal control samples Control procedure of equipment Remarks... 7

3 UCAN-U 0003 Version 1 November 2010 Page 3 of 7 1. Subject This standard operating procedure (SOP) describes a procedure for multicolor immunofluorescent staining with antibodies against intracellular cytokines and cell surface markers of cells within mixed cell populations. 2. Application Area s 2.1 Application Multicolor immunofluorescent staining with antibodies of individual cells (PBMC, SFMC) 2.2 Normal Values N/A 2.3 Minimum and maximum values N/A 2.4 Protocols which follow up this assay Procedure for Setting Compensation for Multi-Color Flow Cytometric Analysis 3. Definitions, Terms and Abbreviations SOP = Standard Operation Procedure PBMC = Peripheral Blood Mononuclear Cells SFMC = Synovial Fluid Mononuclear Cells FACS = Fluorescence Ativated Cell Sorter PBS = Phosphate Buffered Saline FBS = Foetal Bovine Serum P/S/G = Penicillin-Streptomycin L-Glutamine RPM = Rotations per Minute 4. Principle Flow cytometry is a powerful analytical technique in which individual cells can be simultaneously analyzed for several parameters, including size and granularity, as well as the expression of surface and intracellular markers defined by fluorescent antibodies. Multicolor immunofluorescent staining with antibodies against intracellular cytokines and cell surface markers provides a high resolution method to identify the nature and frequency of cells which express a particular cytokine and surface markers. The individual cells can be characterized and separated in a machine called a fluorescenceactivated cell sorter (FACS) that measures cell size, granularity and fluorescence due to bound fluorescent antibodies as single cells pass in a stream past photodetectors. The analysis of single cells in this way is called flow cytometry and the instruments that carry out the measurements and/or sort cells are called flow cytometers or cell sorters The addition of GolgiStop to stimulated lymphoid cells blocks their intracellular protein transport processes. This results in the accumulation of most species of cytokine proteins in the

4 UCAN-U 0003 Version 1 November 2010 Page 4 of 7 Golgi complex. The increased accumulation of cytokine proteins in cells thus enhances the detectability of cytokine-producing cells with immunofluorescent staining and flow cytometric analysis. To block the Fc receptors mouse serum is added to the cells before incubation with the fluorochrome-conjugated monoclonal antibodies specific for the cell surface antigens. Some antibodies which recognize native cell surface markers may not bind to fixed/denatured antigen. For this reason, it is recommended that the staining of cell surface antigens be done with live, unfixed cells prior to fixation/permeabilization and stainig of intracellular cytokines 5. Safety precautions Treed every sample containing human material such as AB serum and the lymphocyte samples as infectious material. GolgiStop Xn, F Cytofix/Cytoperm Xn Sodium azide T+, N Paraformaldehyde T 6. Reagents 6.1 Chemicals Reagents Formula Supplier Order number Store Cytofix/Cytoperm BD Pharmingen C Foetal Bovine Serum (FBS) Invitrogen C GolgiStop BD Pharmingen C L-Glutamine 200MM nvitrogen C Normal mouse serum Rockland D208-00/ C Penicillin-Streptomycin nvitrogen C Perm/Wash BD Pharmingen C RPMI 1640 Medium nvitrogen C Sodium azide NaN 3 Sigma S-2002 RT Paraformaldehyde (CH 2 O) n Fluka RT 6.2 Facs Buffer (PBS with 2% FCS and 0.1% Sodium Azide) 980 ml PBS 20 ml FBS 1 gram Sodium Azide Exp.Date : 3 months at +4 C 6.3 GolgiStop (Pre-dilution) First make a pre-dilution of GolgiStop (1:100) in Culture Medium. Then add for example 67 µl to 1.0 ml of PBMC s or 13.3 µl to 200 µl of PBMC s Exp. Date : Add immediately after preparation

5 UCAN-U 0003 Version 1 November 2010 Page 5 of Culture Medium (RPMI with P/S/G and 2.0% FCS) 0.5 Litre RPMI 1640 medium 5 ml P/S (one tube) 5 ml Glu (one tube) 10 ml FBS (filtered through a 0.20 µm filter) Exp.Date : 1 month at +4 C 6.5 Fixation buffer (PBS + 1.0% Paraformaldehyde) 1 Litre PBS 10 gram Paraformaldehyde Exp.Date : 6 months at +4 C 7. Equipment and Accessories tools 7.1 Equipment Centrifuge, Heraeus Megafuge 1.0R Centrifuge, Hettich Rotanta 46 Plate Shaker, IKA MS2 Minishaker Plate Shaker, Heidolph Titramax 101 Vortex, Janke & Kunkel VF2 BD FACSVantage SE BD FACSCalibur 7.2 Accessories 96 round bottom wells plate, Nunc Roskilde, Cat.No Nunc Micronic tubes Standard 1.4 ml polypropylene tubes, Micronic, Cat. No. M32000 Nunc Micronic Traxis polypropylene rack with cover, Micronic, Cat.No. M42000 Nunc Micronic Capband-8 polypropylene, Micronic, Cat.No. M227C2 Falcon 5 ml Polystyrene Round-bottom tubes, Becton Dickinson, Cat.No Multichannel Pipet, Labsystems Finnpipette, 5 50 µl Multichannel Pipet, Labsystems Finnpipette, µl 8. Samples 8.1 Sample Collection See SOP No. 8.2 Sample Processing Immunofluorescent staining is performed on fresh/thawed PBMC s and SFMC s. 9. Procedure Treatment of stimulated cells for 4 to 6 hours with GolgiStop significantly increases the ability to detect cytokine-producing cells by immunofluorescent staining. Add the pre-dilution GolgiStop directly in the well/sample (Note: It is recommended that GolgiStop not be kept in cell culture for longer than 12 hours. 1. Leave the cells in the 96 well plate or resuspend the cells of the different conditions from

6 UCAN-U 0003 Version 1 November 2010 Page 6 of 7 the culture plate and collect them in a 5 ml Falcon tube 2. Pellet the cells (1600 rpm, 5 min, +4 C) 3. Discard the supernatant very carefully 4. Wash the cells once by resuspending the pellet in 200 µl Facs buffer (cold) 5. Pellet the cells (1600 rpm, 5 min, +4 C) 6. Discard the supernatant very carefully 7. Resuspend the cells per condition (±2.0x10 5 Cells) in 50 µl Facs buffer 8. Add 1 µl Mouse serum to each well and incubate for 5 min (+4 C) 9. Add the pre-titrated optimal concentration of a fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen to different conditions 10. Incubate 20 min at +4 C in the dark. Place the plates on a plate shaker and shake slowly 11. Wash the cells by adding 150 µl Facs buffer 12. Pellet the cells (1600 rpm, 5 min, +4 C) 13. Discard the supernatant very carefully 14. Wash the cells once by resuspending the pellet in 200 µl Facs buffer (cold) 15. Pellet the cells (1600 rpm, 5 min, +4 C) 16. Discard the supernatant very carefully 17. When also staining for intracellular cytokines continue at step Fix the cells by adding 50 µl Fixation buffer per well 19. Incubate 30 min (or overnight) at +4 C in the dark. Place the plates on a plate shaker and shake slowly 20. Wash the cells by adding 100 µl Facs buffer 21. Pellet the cells (1600 rpm, 5 min, +4 C) 22. Discard the supernatant very carefully and resuspend the pellet in 100 µl of Facs buffer 23. Transfer the samples to the Nunc Micronic tubes containing 150 µl Facs buffer 24. Wash the cells by resuspending the pellet in 200 µl Facs buffer (cold) 25. Pellet the cells (1600 rpm, 5 min, +4 C) 26. Discard the supernatant very carefully 27. Fix the cells by resuspending the pellet in 50 µl Cytofix/Cytoperm per well 28. Incubate 30 min at +4 C in the dark. Place the plates on a plate shaker and shake slowly 29. Wash the cells by adding 100 µl Perm/Wash (make a 1:10 dilution with ddh 2 O) 30. Pellet the cells (1600 rpm, 5 min, +4 C) 31. Discard the supernatant very carefully 32. Wash the cells for a second time by adding 100 µl Perm/Wash buffer 33. Pellet the cells (1600 rpm, 5 min, +4 C) 34. Discard the supernatant very carefully 35. Resuspend the cells per condition (±2.0x10 5 Cells) in 50 µl Perm/Wash buffer 36. Add the pre-titrated optimal concentration of a fluorochrome-conjugated monoclonal antibody specific for a intracellular antigen to different conditions 37. Incubate 30 min at +4 C in the dark. Place the plates on a plate shaker and shake slowly 38. Wash the cells by adding 100 µl Perm/Wash (make a 1:10 dilution with ddh 2 O) 39. Pellet the cells (1600 rpm, 5 min, +4 C) 40. Discard the supernatant very carefully 41. Wash the cells for a second time by adding 100 µl Perm/Wash buffer 42. Pellet the cells (1600 rpm, 5 min, +4 C) 43. Discard the supernatant very carefully 44. Resuspend the pellet in 100 µl Facs buffer 45. Transfer the samples to the Nunc Micronic tubes containing 150 µl Facs buffer

7 UCAN-U 0003 Version 1 November 2010 Page 7 of Documentation Save the files after analysis on the FACS digitally in FacsDiva and make a note in your labjournal 11. Quality Control 11.1 Internal control samples. Iso-type controls can be used to discriminate specific staining from artifactual staining. Investigators should choose which staining controls best meet their research needs Control procedure of equipment The FacsCanto is checked twice a year by BD Biosciences and CST performance check is performed each week. 12. Remarks Cells that are surface stained only and acquired immediately or within a few hours, do not need to be fixed Fixed surface stained cells can be kept at +4 C in the dark, using aluminium foil, for weeks Fixed intracellular stained cells can be kept at +4 C in the dark, preferably for no longer than 1 week. Though cytokines should not leak out of the cells after fixation, experiences differ with the amount of cytokines left after a period of time Keep the antibodies at +4 C at all times. Keep once out of the fridge the antibodies on ice during staining. *** End ***