IL-2 Secretion Assay IL-2 Secretion Assay
Contents
1. Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 83060 Fax 49 2204 85197 e mail macs miltenyibiotec de Miltenyi Biotec Inc 12740 Earhart Avenue Auburn CA 95602 USA Phone 800 FOR MACS 530 888 8871 Fax 530 888 8925 e mail macs miltenyibiotec com Miltenyi Biotec Ltd UK Phone 01483 799 800 Fax 01483 799 811 e mail macs miltenyibiotec co uk MACS Cytokine Secretion Assays Miltenyi Biotec France Phone 01 56 98 16 16 Fax 0156981617 e mail macs miltenyibiotec fr Miltenyi Biotec S r l Italy Phone 051 64 60 411 Fax 0516460499 e mail macs miltenyibiotec it Miltenyi Biotec S L Spain Phone 91 512 12 90 Fax 915121291 e mail macs miltenyibiotec es Miltenyi Biotec GmbH Shanghai Office Phone 021 58305365 Fax 021 58305243 e mail miltenyibiotec china com For further information refer to our website www miltenyibiotec com For technical questions please contact your local distributor or our Technical Support Team in Germany e mail macsTec miltenyibiotec de phone 49 2204 830 6 830 Miltenyi Biotec v0 26S 000 07 L MACS This MACS product is for in vitro research use only and not for diagnostic or therapeutic procedures 1 Description 1 1 Principle of the IL 2 Secretion Assay 1 2 Background and product applications 1 3 Reagent and instrument requirements 2 Protocol overview 3 Experimental set up 3 1 C2. before enrichment after enrichment O 3 O 3 Bi Pe m A O 0 4 li anti IL 2 PE anti IL 2 PE 0 004 of the total CD4 T cell population 1 IL 2 CD4 T cell were enriched from 10 secrete IL 2 CD4 cells lt 0 0001 MAGS 140 000 592 04 24 1 Manz R Assenmacher M Pfl ger E Miltenyi S Radbruch A 1995 Analysis and A z Sorting of Live cells According to Secreted Molecules Relocated to a Cell Surface A Flask and dish sizes for stimulation Affinity Matrix Proc Natl Acad Sci USA 92 1921 1925 139 7 Appendix For in vitro stimulation see 4 2 step 2 the cells should be resuspended in culture 2 Assenmacher M Lohning M Scheffold A Manz RA Schmitz J Radbruch A medium containing 5 of human serum at 10 cells ml and 5x10 cells cm Both the 1998 Sequential production of IL 2 IFN y and IL 10 by individual staphylococcal dilution and the cell density are important to assure optimum stimulation enterotoxin B activated T helper lymphocytes Eur J Immunol 28 1534 1543 The following table lists culture plate dish and flask sizes suitable for different cell 483 numbers It also indicates the appropriate amount of medium to add 3 Brosterhus H Brings S Leyendeckers H Manz RA Miltenyi S Radbruch A Assenmacher M Schmitz J 1999 Enrichment and detection of live antigen total cell medium volume culture well specific CD4 and CD8 T ce
3. of the cell enrichment To illustrate the analysis we describe the detection of IL 2 secreting T cells using the IL 2 Secretion Assay The detailed description including how to set gates should serve as a model for the analysis of your own sample Miltenyi Biotec 21 140 000 592 04 A Lymphocyte gate in the forward versus side scatter plot before enrichment after enrichment side scatter side scatter forward scatter forward scatter B Dead cell and monocyte exclusion in FL 2 versus FL 3 before enrichment after enrichment propidium iodide CD14PerCP propidium iodide CD14PerCP rir rrm R E anti IL 2 PE anti IL 2 PE Miltenyi Biotec 23 140 000 592 04 1 10 human PBMC of a CMV donor have been restimulated for 16 hours with and without CMV lysate 5 ug ml Biowhittaker 2 The IL 2 Secretion Assay was performed on the stimulated and the unstimulated sample 3 Counterstaining of T cells was performed using CD4 FITC 4 Monocytes were stained with CD14 PerCP Dead cells were stained with propidium iodide PI which was added just prior to flow cytometric analysis to a final concentration of 0 5 ug ml 6 200 000 viable cells of the fractions before enrichment and the complete enriched fractions were acquired by flow cytometry from the stimulated and the unstimulated samples 7 A lymphocyte gate based on forward and side scatter FSC SSC properties was activated prior to
4. Separators Column max number max number Separator of labeled cells of total cells MS 10 2x 108 MiniMACS OctoMACS with Column Adapter VarioM ACS SuperMACS LS 108 2x10 MidiMACS with Column Adapter VarioMACS SuperM ACS autoMACS 2x108 4x10 autoM ACS A Note Column adapters are required to insert certain columns into VarioMACS or SuperMACS For details see MACS Separator data sheets Refrigerated centrifuge 4 8 C e Rotation device for tubes MACSmix 130 090 753 Optional Pre Separation Filter 130 041 407 140 000 592 04 8 2 Protocol overview A Cell preparation see 4 1 Whole blood PBMC cell culture or tissue preparation A B Antigen specific In vitro stimulation see 4 2 antigen sample control sample incubation with incubation without antigen antigen lt 3 16 hours E 3C C IL 2 Secretion Assay see 4 3 e Labeling with IL 2 Catch Reagent 5 minutes on ice e IL 2 secretion period 45 minutes 37 C e Labeling with IL 2 Detection Antibody 10 minutes on ice e Magnetic labeling with Anti PE MicroBeads see 4 4 15 minutes 4 8 C D Magnetic Separation see 4 5 1 over 2 MS or LS Columns t or with the E autoMACS E E E Detection analysis see 5 cell culture or subsequent experiment Miltenyi Biotec 9 140 000 592 04 Peptide is then added the next morning for 3 hours of stimulation directly follo
5. further gating to exclude monocytes and debris see A 8 Dead cells and monocytes were excluded according to PI and CD14 PerCP staining in a fluorescence 2 PE versus fluorescence 3 plot PerCP see B The dead cell exclusion is crucial for the analysis of rare antigen specific T cells as dead cells may bind non specifically to antibodies or MicroBeads This could lead to false positive events The sensitivity of detection is further enhanced by exclusion of undesired non T cells like monocytes which may cause non specific background staining 9 Analysis of secreted IL 2 PE versus CD4 FITC staining by viable lymphocytes is displayed see C MAGS 140 000 592 04 22 C Antigen specific CD4 T cells stained for secreted IL 2 Sample stimulated with CMV lysate before enrichment after enrichment CD4 FITC CD4 FITC anti IL 2 PE anti I L 2 PE 0 350 of the total CD4 T cell population The IL 2 secreting CD4 T cells have been secrete IL 2 see formula below enriched to 80 4 2047 IL 2 CD4 T cells were enriched from 10 CD4 cells 0 205 see formula below IL 2 cells among CD4 of IL 2 CD4 cells in the analyzed sample of total CD4 cells in the analyzed sample IL 2 cells among CD4 abs of IL 2 CD4 cells in the enriched fraction abs of total CD4 cells before enrichment x 10 x 100 Unstimulated control sample
6. highest sensitivity of analysis 1 2 Background and product applications The IL 2 Secretion Assay Cell Enrichment and Detection Kit is designed for the detection isolation and analysis of viable IL 2 secreting leukocytes It is specially developed for the detection and isolation of antigen specific T cells After restimulation with specific antigen in vitro secretion of IL 2 is induced IL 2 is rapidly secreted by naive T helper cells and by certain subsets of memory T cells upon activation It promotes growth and differentiation of T cells and has pleiotropic effects on many other leukocytes Quantitative analysis of antigen specific T cell populations can provide important information on the natural course of immune responses MACS enrichment of the antigen specific T cells increases the sensitivity of analysis allowing detection of frequencies as low as one in a million cells The MACS enrichment also enables further functional characterization of the antigen specific cells and downstream experiments as well as the expansion of antigen specific cells allowing research on potential future immunotherapeutical applications Examples of applications Detection and enrichment of viable IL 2 secreting leukocytes Detection and enrichment of IL 2 secreting antigen specific T cells for enumeration and phenotypic analysis as well as for expansion and functional characterization MAGS 140 000 592 04 6 MACS Columns and MACS
7. of erythrocyte lysing solution room temperature A Work fast keep the cells cold use pre cooled solutions which will prevent capping of antibodies on the cell surface and a non specific cell labeling exception warm medium during secretion period and room temperature during lysing step A Do not remove supernatant by decanting This will lead to cell loss and incorrect incubation volumes Pipette off or aspirate supernatant A Dead cells may bind non specifically to MACS MicroBeads or antibodies Therefore when working with cell preparations containing large amounts of dead cells they should be removed before starting the Cytokine Secretion Assay e g by density gradient centrifugation or by using the Dead Cell Removal Kit 130 090 101 A Higher temperatures and longer incubation times for staining should be avoided This will lead to non specific cell labeling Lysis of erythrocytes 1 After stimulation add 45 ml of erythrocyte lysing solution to 5 ml whole blood sample 2 Mix gently and incubate for 10 minutes at room temperature Rotate tube continuously using the MACSmix 130 090 753 or turn tube several times during incubation Miltenyi Biotec 31 140 000 592 04 A Do not use media containing any non human proteins like BSA or FCS because of non specific stimulation See Protocol for in vitro stimulation 1 Start with 5 ml of fresh sodium heparinized human blood containing about 10 lymphocytes
8. BSA and 2 mM EDTA e g 4 ml of a 0 5 M EDTA stock solution per 1 liter of buffer Miltenyi Biotec 27 140 000 592 04 140 000 592 04 28 MACS Columns and MACS Separators Column max number max number Separator of labeled cells of total cells MS 10 2x108 MiniMACS OctoMACS with Column Adapter VarioMACS SuperMACS autoMACS 2x10 4x10 autoMACS A Note Column adapters are required to insert certain columns into VarioMACS or SuperMACS For details see MACS Separator data sheets Optional Rotation device for tubes MACSmix 130 090 753 Optional Pre Separation Filter 130 041 407 B2 Protocol B2 1 Antigen specific in vitro stimulation A The peripheral blood should not be older than 20 hours and should be supplemented with anticoagulant sodium heparin Do not use EDTA or ACD Lymphocyte activation and secretion of cytokines requires calcium and is consequently inhibited by chelating anticoagulants A Note Whole blood may be stored overnight at room temperature A Always include a negative control sample in the experiment A positive control with e g Staphylococcal Enterotoxin B SEB may be included in the experiment see also detailed protocol provided with the Cytokine Secretion Assay Kits Miltenyi Biotec 29 140 000 592 04 A For each sample with 5 ml whole blood prepare 100 ml of cold buffer 4 8 C 200 ul of cold medium 4 8 C 7 ml of warm medium 37 C 45 ml
9. Catch Reagent per 10 total cells mix well and incubate for 5 minutes on ice 140 000 592 04 16 IL 2 secretion period 1 Add warm 37 C medium to dilute the cells according to the following table Expected number of Dilution Amount of medium to IL 2 secreting cells add per 10 total cells lt 5 10 cells ml 10 ml gt 5 lt 10 cells ml 100 ml A Note For frequencies of cytokine secreting cells gt gt 20 the cells need to be further diluted e g by a factor of 5 2 Incubate cells in closed tube for 45 minutes at 37 C under slow continuous rotation using the MACSmix 130 090 753 or turn tube every 5 minutes to resuspend settled cells A Note During this step it is crucial to prevent contact of cells to avoid cross contamination with cytokines Labeling cells with IL 2 Detection Antibody Put the tube on ice 1 2 Wash the cells by filling up the tube with cold buffer and centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely A Note If the volume of the cell suspension was higher than the volume of added buffer repeat wash step 3 Resuspend cell pellet in 80 ul of cold buffer per 10 total cells Miltenyi Biotec 17 140 000 592 04 4 5 Magnetic separation Magnetic separation using MS or LS Columns A Choose an appropriate MACS Column and MACS Separator according to the number of total cells see table in 1 3 A When enriching antige
10. HCO 100 mM of the Cytokine Secretion Assay Cell Enrichment and Detection 1 ml 0 5 M EDTA 1 mM adjust pH to 7 3 fill up to 500 ml with Kits for human cells dd H O B 1 Reagent and instrument requirements A Note Do not use FACS Lysing solution e Optional Staining reagents CD4 FITC 130 080 501 or CD8 FITC 130 080 601 and CD14 PerCP A Note Do not use tandem conjugates of phycoerythrin like Cy Chrome Cytokine Secretion Assay Kit for example IFN y Secretion Assay Cell Enrichment and Detection Kit PE 130 054 201 PharMingen PE Cy5 Serotec ECD PC5 Coulter Immunotech etc they IL 2 Secretion Assay Cell Enrichment and D merece a may also be recognized by the Anti PE MicroBeads f A Note U tivati f T cells TCR and iated molecules lik IL 4 Secretion Assay Cell Enrichment and Detection Kit PE o oe a va ae 4 Sn a ye erst De 130 054 101 MIS e down regulated IL 10 Secretion Assay Cell Enrichment and Detection Kit PE A Note For optimal sensitivity we recommend labeling of undesired non T cells such 130 090 43 5 as monocytes with antibodies conjugated to PerCP e g CD14 PerCP These cells can then be excluded together with PI stained dead cells by gating Propidium iodide PI or 7 AAD to exclude dead cells from analysis Anticoagulant sodium heparin e Buffer degassed phosphate buffered saline pH 7 2 containing 0 5 bovine serum albumin
11. Resuspend cells in culture medium containing 5 human serum adjust to 10 cells ml and 5x10 cells cm see 7 Appendix A Flask and dish sizes for stimulation 3 Add antigen or control reagent peptide 3 6 hours at 37 C 5 7 CO e g 1 10 ug ml protein 6 16 hours at 37 C 5 7 CO e g 10 ug ml SEB 3 16 hours at 37 C 5 7 CO e g 1 ug ml For comparison of different experiments the stimulation time should always be the same see 3 2 4 Collect cells carefully by using a cell scraper or by pipetting up and down when working with smaller volumes Rinse the dish with cold buffer Check microscopically for any remaining cells if necessary rinse the dish again 140 000 592 04 14 large amounts of dead cells they should be removed before starting the IL 2 Secretion Assay e g by density gradient centrifugation or by using the Dead Cell Removal Kit 130 090 101 Labeling cells with IL 2 Catch Reagent 1 Use 10 total cells in a 15 ml closable tube per sample A Note For larger cell numbers scale up all volumes accordingly For less than 10 cells use same volumes 2 Wash cells by adding 10 ml of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C pipette off supernatant completely A Note Do not remove supernatant by decanting This will lead to cell loss and incorrect incubation volumes 3 Resuspend cell pellet in 80 ul of cold medium per 10 total cells 4 Add 20 ul of IL 2
12. by flow cytometry the medium should not contain phenol red 11 Proceed to analysis see section 5 cell culture or other subsequent experiment Magnetic separation using the autoMACS A Refer to the auttoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACs 140 000 592 04 20 2 Optional Pass cells through Pre Separation Filter 130 041 407 to remove clumps 3 Place tube containing magnetically labeled cells in autoMACS Choose separation program Posseld Collect the separated fractions from outlet port pos2 4 Proceed to analysis see section 5 cell culture or other subsequent experiment 5 Detection and analysis of IL 2 secreting T cells A Add propidium iodide PI or 7 AAD to a final concentration of 0 5 ug ml just prior to acquisition for exclusion of dead cells from flow cytometric analysis Incubating with PI for longer periods will affect the viability of the cells Do not fix the cells when using PI or 7 AAD A For optimized sensitivity an appropriate number of viable cells has to be acquired from the antigen stimulated sample as well as from the control sample Acquire 2x10 viable cells from the fraction before enrichment see 4 4 step 5 For enumeration of low frequent IL 2 secreting cells acquire all of the positive fraction For preparative purposes acquire an aliquot of the positive fraction to determine the performance
13. creting cells The cells can now be used for cell culture or analysis Since viable Miltenyi Biotec gt 140 000 592 04 Monitoring and analysis of antigen specific T cell immunity e g in infection autoimmunity cancer allergy or alloreactivity Isolation and expansion of antigen specific T cells for research in immunotherapy Enrichment and analysis of IL 2 secreting cells for determination of functional antigens in disease and for T cell receptor TCR epitope mapping Analysis or cloning of TCR repertoire of antigen specific T cells 1 3 Reagent and instrument requirements Buffer degassed phosphate buffered saline pH 7 2 containing 0 5 bovine serum albumin BSA and 2 mM EDTA e g 4 ml of a 0 5 M EDTA stock solution per 1 liter of buffer Optional 0 5 M EDTA stock solution dissolve 56 g sodium hydroxide NaOH in 900 ml dd H O Add 146 2 g ethylene diamine tetraacetic acid EDTA adjust pH to 7 5 fill up to 1000 ml with dd H O Culture medium e g RPMI 1640 containing 5 human serum like autologous or AB serum do not use BSA or FCS because of non specific stimulation Propidium iodide PI or 7 AAD to exclude dead cells from analysis Optional Staining reagents such as CD4 FITC 130 080 501 or CD8 FITC 130 080 601 and CD14 PerCP Miltenyi Biotec 7 140 000 592 04 cells are analyzed non specific background can be minimized by dead cell exclusion This provides
14. e Storage Store protected from light at 4 8 C Do not freeze The expiration dates are indicated on the vial labels 1 1 Principle of the IL 2 Secretion Assay Antigen specific T cells are analyzed and isolated using the IL 2 Secretion Assay starting from whole blood PBMC or other leukocyte containing single cell preparations The cells are restimulated for a short period of time with specific peptide protein or other antigen preparations 140 000 592 04 4 Anti PE MicroBeads IL 2 Catch A Reagent IL 2 Detection Antibody PE N IL 2 secreting cell Subsequently an IL 2 specific Catch Reagent is attached to the cell surface of all leukocytes The cells are then incubated for a short time at 37 C to allow cytokine secretion The secreted IL 2 binds to the IL 2 Catch Reagent on the positive secreting cells These cells are subsequently labeled with a second IL 2 specific antibody the IL 2 Detection Antibody conjugated to phycoerythrin PE for sensitive detection by flow cytometry The IL 2 secreting cells can now be magnetically labeled with Anti PE MicroBeads and enriched over a MACS Column which is placed in the magnetic field of a MACS Separator The magnetically labeled cells are retained in the MACS Column while the unlabeled cells run through After the column has been removed from the magnetic field the magnetically retained cells can be eluted as positively selected cell fraction enriched for cytokine se
15. e at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely Miltenyi Biotec 33 140 000 592 04 7 Pipette 1 ml of cold buffer on top of the first column Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column directly onto the second column 8 Collect unlabeled cells that pass through and wash with 3x500 ul of cold buffer Perform washing steps by adding buffer successsively once the column reservoir is empty 9 Remove second column from separator place column on a suitable collection tube 10 Pipette 500 ul of cold buffer on top of the column Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column A Note For subsequent cell culture the cells can also be eluted with medium If part of the cells are analysed by flow cytometry the medium should not contain phenol red 11 Proceed to flow cytometric analysis see detailed protocol cell culture or other subsequent experiment Magnetic separation using the autoMACS A Refer to the auttoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACs 2 Optional Pass cells through Pre Separation Filter 130 041 407 to remove clumps Miltenyi Biotec 35 140 000 592 04 4 Resuspend cell pellet in 500 ul of cold buffer 5 Optional Take an aliquot for flow cytomet
16. icroBeads per 10 total cells mix well and incubate for 15 minutes at 4 8 C A Note Incubate in refrigerator at 4 8 C do not work on ice during this step 3 Wash cells by adding 10 ml of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant 4 Resuspend cell pellet in 500 ul of cold buffer For higher cell numbers than 5x10 use a dilution of 10 cells ml 5 Optional Take an aliquot for flow cytometric analysis and cell count of the fraction before enrichment 6 Proceed to magnetic separation see 4 5 140 000 592 04 18 7 Pipette appropriate amount of cold buffer onto the first column Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column directly onto the second column MS 1 ml LS 5 ml 8 Collect unlabeled cells that pass through and wash with appropriate amount of cold buffer Perform washing steps by adding buffer successively once the column reservoir is empty MS 3x500 ul LS 3x3 ml 9 Remove the second column from separator place the column on a suitable collection tube 10 Pipette appropriate amount of cold buffer onto the column Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column MS 500 ul LS 5 ml A Note For subsequent cell culture the cells can also be eluted with medium If part of the cells are analyzed
17. in a 50ml conical polypropylene tube 2 Add the antigen or as a positive control 1 ug ml SEB for 3 16 hours at 37 C 5 7 CO for details on the kinetics of cytokine secretion and on concentrations of antigen to add refer to Cytokine Secretion Assay data sheet 3 1 3 2 3 A negative control sample treated exactly the same as the antigen stimulated sample but without addition of antigen should always be included in the experiment 4 Optional Costimulatory agents like CD28 and CD49d antibodies may be added B2 2 Cytokine Secretion Assay A This protocol is optimized for cell samples containing lt 5 of total cytokine secreting cells If gt 5 of cytokine secreting cells are expected it is necessary to dilute the cells further during the cytokine secretion period and therefore a larger test tube will be needed The dilution avoids non specific staining of cells not secreting cytokines during this period A 140 000 592 04 30 3 Centrifuge cells at 300xg for 10 minutes at room temperature remove supernatant completely Labeling cells with Cytokine Catch Reagent 1 Resuspend cell pellet in 15 ml of cold buffer and transfer into a 15 ml conical propylene tube 2 Centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely 3 Resuspend pellet in 160 ul of cold medium 4 Add 40 ul of Cytokine Catch Reagent mix well and incubate for 5 minutes on ice Le Cytokine secretion pe
18. lls based on cytokine secretion Eur J Immunol 29 number to add plate diameter 4053 4059 573 0 15 x 107 0 15 ml 96 well 0 64 cm 4 Oelke M Moehrle U Chen JL Behringer D Cerundolo V Lindemann A ore ae tee eae a een 0 5 x 10 0 5 ml 48 well 1 13 Mackensen A 2000 Generation and purification of CD8 Melan A Specific ii iii Cytotoxic T Lymphocytes for Adoptive Transfer in Tumor Immunotherapy Clin 1x10 1ml 24 well 1 6 cm Cancer Res 6 1997 2005 663 2x10 2 ml 12 well 2 26 cm 5 Oelke M Kurokawa T Hentrich I Behringer D Cerundolo V Lindemann 5x10 5 ml 6 well 3 5 cm A Mackensen A 2000 Functional Characterization of CD8 Antigen Specific Cytotoxic T Lymphocytes after Enrichment Based on Cytokine Secretion total cell di it dish Comparison with the MHC Tetramer Technology Scand J Immunol 52 544 549 seen eee oga cr ge 970 l l 4 5 x 10 4 5 ml small 3 5 cm 6 Bickham K Miinz C Tsang ML Larsson M Fonteneau J F Bhardwaj N Steinmann R 2001 EBNA1 specific CD4 T cells in healthy carriers of Epstein 10 x 10 10 ml medium 6 cm Barr virus are primarily Th1 in function J Clin Invest 107 121 130 1035 25x10 25 ml large 10 cm 7 Pittet MJ Zippelius A Speiser DE Assenmacher M Guillaume P Valmori D 50 x 10 50 ml extra large 15 cm Lienard D Lejeune F Cerottini JC Romero P 2001 Ex vivo IFN y secretion by circulating CD8 T lymphocytes Implications of a no
19. lyzed simultaneously for IFN y or IL 10 production by two color cytokine analysis combining the IL 2 Secretion Assay with the IFN y Secretion Assay Detection Kit APC 130 090 762 or the IL 10 Secretion Assay Detection Kit APC 130 090 761 Detailed protocols are included in the data sheets of the Cytokine Secretion Assay Detection Kits APC and are available from our website www miltenyibiotec com 3 5 Combination with peptide MHC tetramer staining IL 2 secreting cells can be analyzed simultaneously for peptide MHC tetramer staining by combining the IL 2 Secretion Assay PE with APC conjugated peptide MHC tetramers For combination with PE conjugated peptide MHC tetramers the IL 2 Secretion Assay Detection Kit APC 130 090 763 is available Detailed recommendations for the experimental setup and the procedure are included in the data sheets of the Cytokine Secretion Assay Detection Kits APC and are available from our website www miltenyibiotec com 140 000 592 04 12 3 6 Detection without prior enrichment Optional If the sample contains more than 0 01 0 1 of IL 2 secreting cells you may be able to analyze IL 2 secreting cells without prior enrichment see also IL 2 Secretion Assay Detection Kit PE 130 090 487 The assay can also be performed directly starting from whole blood A detailed protocol is included in the data sheet of the IL 2 Secretion Assay Detection Kit PE and is available from
20. n specific T cells always perform two consecutive column runs to achieve best results 1 Prepare two columns per sample by rinsing with cold buffer MS 500 ul LS Column 3 ml and discard effluent 2 Place the first column into the magnetic field of a MACS Separator use column adapter with VarioMACS or SuperMACS 3 Optional Pass the cells through Pre Separation Filter 130 041 407 to remove clumps 4 Apply cell suspension onto the column 5 Collect unlabeled cells which pass through and wash with appropriate amount of cold buffer Perform washing steps by adding buffer successively once the column reservoir is empty MS 3x500 ul LS 3x3 ml Collect total effluent This is the unlabeled cell fraction 6 Remove the first column from separator place the second column into the separator and put the first column on top of the second one Miltenyi Biotec 19 140 000 592 04 4 Add 20 ul of IL 2 Detection Antibody PE per 10 total cells 5 Optional Add additional staining reagents e g 10 ul of CD4 FITC 130 080 501 or 10 ul of CD8 FITC 130 080 601 and CD14 PerCP 6 Mix well and incubate for 10 minutes on ice 7 Wash cells by adding 10 ml of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C pipette off supernatant 4 4 Magnetic labeling Magnetic labeling with Anti PE MicroBeads Resuspend cell pellet in 80 ul of cold buffer per 10 total cells 1 2 Add 20 ul of Anti PE M
21. ontrols 3 2 Kinetics of restimulation and proposed time schedule 3 3 Counterstaining of cytokine secreting cells 3 4 Two color cytokine analysis 3 5 Combination with peptide MHC tetramer staining 3 6 Detection without prior enrichment 4 Protocol for the IL 2 Secretion Assay 4 1 Cell preparation 4 2 Antigen specific In vitro stimulation 4 3 Cytokine Secretion Assay 4 4 Magnetic labeling 4 5 Magnetic separation 5 Detection and analysis of IL 2 secreting antigen specific T cells 6 References Appendix A Flask and dish sizes for stimulation B Detection and enrichment of cytokine secreting cells from whole blood Miltenyi Biotec 3 140 000 592 04 MACS Cytokine Secretion Assays IL 2 Secretion Assay Cell Enrichment and Detection Kit PE human Order No 130 090 488 7 oy For 50 tests with 10 cells T N Miltenyi Biotec MAGS 1 Description Components 1 ml IL 2 Catch Reagent anti IL 2 monoclonal antibody mouse IgG1 conjugated to cell surface specific monoclonal antibody mouse IgG2a 1 ml IL 2 Detection Antibody anti IL 2 monoclonal antibody mouse IgG2a conjugated to PE phycoerythrin 1 ml Anti PE MicroBeads colloidal super paramagnetic MicroBeads conjugated to monoclonal mouse anti PE antibody mouse IgG1 Size For 50 tests with 10 cells Product format All components are supplied as a suspension containing 0 1 gelatine and 0 05 sodium azid
22. our website www miltenyibiotec com 4 Protocol for the IL 2 Secretion Assay For the detection and isolation of cytokine secreting cells best results are achieved by starting the assay with fresh PBMC or other leukocyte containing single cell preparations from tissues or cell lines Alternatively frozen cell preparations can be used A Note PBMC may be stored over night The cells should be resuspended and incubated in culture medium as described in 4 2 step 2 but without addition of antigen The antigen is then added to the culture on the next day A Note Remove platelets after density gradient separation Resuspend cell pellet fill tube with buffer and mix Centrifuge at 200xg for 10 15 minutes at 20 C Carefully remove supernatant Special protocols for whole blood You can start the IL 2 Secretion Assay directly from whole blood For details on the procedure see 7 Appendix B Detection and enrichment of cytokine secreting cells from human whole blood This special protocol is also available from our website www miltenyibiotec com Miltenyi Biotec 13 140 000 592 04 4 3 Cytokine Secretion Assay General considerations A The assay is optimized for cell samples containing lt 5 of total IL 2 secreting cells If gt 5 of IL 2 secreting cells are expected it is necessary to dilute the cells further during the cytokine secretion period and therefore a larger test tube will be needed see table below The dilution prevent
23. ric analysis and cell count of the fraction before enrichment 6 Proceed to magnetic separation B2 4 Magnetic separation Magnetic separation using MS Columns A When enriching antigen specific T cells always perform two consecutive MS Columns to achieve best results 1 Prepare two MS Columns per sample by rinsing with 500 ul cold buffer discard effluent 2 Place first column into the magnetic field of a MACS Separator use column adapter with VarioMACS or SuperMACS 3 Optional Pass cells through Pre Separation Filter 130 041 407 to remove clumps 4 Apply cell suspension onto the column 5 Collect unlabeled cells which pass through and wash with 3x500 ul of cold buffer Perform washing steps by adding buffer successively once the column reservoir is empty Collect total effluent This is the unlabeled cell fraction 6 Remove first column from separator place second column into the separator and put the first column on top of the second one MAGS 140 000 592 04 34 3 Place tube containing magnetically labeled cells in autoMACS Choose separation program Posseld Collect the separated fractions from outlet port pos2 4 Proceed to flow cytometric analysis see detailed protocol cell culture or other subsequent experiment Warning Reagents contain sodium azide Sodium azide yields hydrazoic acid under acid conditions which is extremely toxic Azide compounds should be diluted with
24. riod 1 Add 7 ml of warm medium 37 C to dilute the cells A Note For frequencies of cytokine secreting cells gt 5 the cells need to be further diluted e g by a factor of 5 2 Incubate cells in a closed tube for 45 minutes at 37 C under slow continuous rotation using the MACSmix or turn tube every 5 minutes to resuspend settled cells A Note During this step it is crucial to prevent contact of cells to avoid cross contamination with cytokines 140 000 592 04 32 Labeling cells with Cytokine Detection Antibody 1 Put the tube on ice 2 Wash cells by adding 8 ml of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely 3 Resuspend cell pellet in 160 ul of cold buffer 4 Add 40 ul of Cytokine Detection Antibody PE 5 Optional Add additional staining reagents e g 20 ul of CD4 FITC 130 080 501 or CD8 FITC 130 080 601 and CD14 PerCP 6 Mix well and incubate for 10 minutes on ice 7 Wash cells by adding 10 ml of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely B2 3 Magnetic labeling Magnetic labeling with Anti PE MicroBeads Resuspend cell pellet in 160 ul of cold buffer 1 2 Add 40 ul of Anti PE MicroBeads mix well and incubate for 15 minutes at 4 8 C A Note Incubate in refrigerator at 4 8 C do not work on ice during this step 3 Wash cells by adding 10 ml of cold buffer centrifug
25. running water before being discarded These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop Warranty Products sold here under are warranted only to be free from defects in workmanship and material at the time of delivery to the customer MILTENYI BIOTEC GmbH makes no warranty or representation either expressed or implied with respect to the fitness of a product for a particular purpose There are no warranties expressed or implied which extend beyond the TeChnical Spesifications of the products MILTENYI BIOTEC GmbH liability is limited to either replacement of the products or refund of the purchase price MILTENYI BIOTEC GmbH is not liable for any property damage personal injury or economic loss caused by the product Cy Chrome is a trademark of PharMingen Peridin Chlorophyll Protein PerCP is a trademark of Becton Dickinson Phycoerythrin PE U S Patent 4 520 110 European Patent 76 695 Australian Patent 548 440 Canadian Patent 1 179 942 Japanese Patent 1 594 827 140 000 592 04 36
26. s non specific staining of cells not secreting IL 2 during this period A For each test with 10 total cells prepare 100 ml of cold buffer 4 8 C 100 ul of cold medium 4 8 C 10 ml or 100 ml see table below of warm medium 37 C A Work fast keep the cells cold use pre cooled solutions which will prevent capping of antibodies on the cell surface and a non specific cell labeling exception warm medium during secretion period A Volumes shown below are for 10 total cells When working with less than 10 cells use the same volumes as indicated When working with higher cell numbers scale up all reagent volumes and total volumes accordingly e g for 2x10 total cells use twice the volume of all indicated reagent volumes and total volumes A Do not remove supernatant by decanting This will lead to cell loss and incorrect incubation volumes Pipette off or aspirate supernatant A Dead cells may bind non specifically to MACS MicroBeads or antibodies Therefore when working with cell preparations containing Miltenyi Biotec 15 140 000 592 04 4 2 Antigen specific In vitro stimulation A Always include a negative control in the experiment A positive control may also be included see 3 1 A Do not use media containing any non human proteins like BSA or FCS because of non specific stimulation ee Protocol for in vitro stimulation 1 Wash cells by adding medium centrifuge at 300xg for 10 minutes 2
27. should always be included This will provide information about IL 2 secretion unrelated to the specific antigen stimulation but e g due to ongoing in vivo immune responses The control sample should be treated exactly the same as the antigen stimulated sample except for the addition of antigen or by using a control antigen Positive control When setting up a new experiment it is recommended to include a positive control As a positive control a sample stimulated with the superantigen Staphylococcal Enterotoxin B Sigma 1 ug ml for 3 16 hours may be included in the experiment A Note Mitogens like PHA or PMA Ionomycin are not recommended for stimulation of a positive control as the resulting high frequencies of IL 2 secreting cells do not allow conclusions on the performance e g sensitivity of the IL 2 Secretion Assay 3 2 Kinetics of restimulation and proposed time schedule Peptides Upon stimulation with peptide the cells can be analyzed for IL 2 secretion 3 6 hours later It is possible to prepare the cells first and take them into culture overnight but without adding the antigen see 4 2 step 2 MAGS 140 000 592 04 10 A For optimal sensitivity we recommend labeling of undesired non T cells such as monocytes with antibodies conjugated to PerCP e g CD14 PerCP These cells can then be excluded together with PI stained dead cells by gating 3 4 Two color cytokine analysis IL 2 secreting cells can be ana
28. vel approach for T cell total cell menn ae rawik monitoring in infectious malignant diseases J Immunol 166 7634 7640 1037 Hura to add flask 5 ated 8 Becker C Pohla H Frankenberger F Sch ler T Assenmacher M Schendel 12x10 12 ml 50 ml 25 cm DJ Blankenstein T 2001 Adoptive tumor therapy with T lymphocytes enriched i through an IFN y capture assay Nature Medicine 7 10 1159 1162 1207 40 x 10 40 mi 250 ml 75cm 80 x 10 80 ml 720 ml 162 cm For further information visit our website www miltenyibiotec com 120 x 10 120 ml 900 ml 225 cm Miltenyi Biotec 25 140 000 592 04 140 000 592 04 26 B Detection and enrichment of cytokine Optional 0 5 M EDTA stock solution dissolve 56 g sodium secreting cells from whole blood hydroxide NaOH in 900 ml dd H O Add 146 2 g ethylene diamine tetraacetic acid EDTA adjust pH to 7 5 fill up to B1 Reagent and instrument requirements 1000 ml with dd H O O B2 P l a rotoco Culture medium e g RPMI 1640 containing 20 of human B 2 1 Antigen specific in vitro stimulation serum like autologous serum or AB serum B 2 2 Cytokine Secretion Assay A Note Do not use BSA or FCS because of non specific stimulation B2 3 Magnetic labeling e Erythrocyte lysing solution 1x B2 4 Magnetic separation prepare freshly from 10x stock solution The following special protocol can be used in combination with one 10x stock solution 41 4 g NH Cl 1 55 M 5 g K
29. wed by the IL 2 Secretion Assay Proteins Upon stimulation with protein the cells can be analyzed for IL 2 secretion 6 16 hours later It is possible to start the stimulation of the cells late in the afternoon and to perform the IL 2 Secretion Assay the following morning Costimulation The addition of costimulatory agents like CD28 or CD49d antibody may enhance the response to the antigen If costimulatory agents are added to the antigen sample they also have to be included in the control sample 3 3 Counterstaining of cytokine secreting cells The IL 2 secreting cells are stained with PE conjugated IL 2 Detection Antibodies To identify cells of interest counterstaining for T cells with e g CD4 FITC 130 080 501 or CD8 FITC 130 080 601 is important A Do not use tandem conjugates of phycoerythrin like Cy Chrome PharMingen PE Cy5 Serotec ECD PCS Coulter Immunotech etc they may also be recognized by the Anti PE MicroBeads A Upon activation of T cells TCR and some associated molecules like CD3 might be down regulated A The samples should be stained with propidium iodide PI or 7 AAD prior to acquisition to exclude dead cells from analysis This will reduce non specific background staining and increase sensitivity Miltenyi Biotec 11 140 000 592 04 3 Experimental set up 3 1 Controls Negative control For accurate detection of IL 2 secreting antigen specific cells a negative control sample
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