IFN-γ Secretion Assay IFN-γ Secretion Assay
Contents
1. Optional Take an aliquot for flow cytometric analysis and cell count of the fraction before enrichment Proceed to magnetic separation see 4 5 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 3 140 000 274 07 4 Protocol for the IFN y Secretion Assay A When enriching antigen 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 Place the first column into the magnetic field of a MACS Separator use column adapter with VarioMACS or SuperMACS Separator Optional Pass the cells through Pre Separation Filters 130 041 407 to remove clumps Apply cell suspension onto the column 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 Remove the first column from separator place the second column into the separator and put the first column on top of the second one Pipette appropriate amount of cold buffer onto the first column Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger2. E 5 La ae propidium iodide CD14PerCP propidium iodide CD14PerCP anti IFN y PE anti IFN y PE Miltenyi Biotec 24 140 000 274 07 6 References 6 References 1 Manz R Assenmacher M Pfl ger E Miltenyi S Radbruch A 1995 Analysis and Sorting of Live cells According to Secreted Molecules Relocated to a Cell Surface Affinity Matrix Proc Natl Acad Sci USA 92 1921 1925 139 Assenmacher M L hning M Scheffold A Manz RA Schmitz J Radbruch A 1998 Sequential production of IL 2 IFN y and IL 10 by individual staphylococcal enterotoxin B activated T helper lymphocytes Eur J Immunol 28 1534 1543 483 Brosterhus H Brings S Leyendeckers H Manz RA Miltenyi S Radbruch A Assenmacher M Schmitz J 1999 Enrichment and detection of live antigen specific CD4 and CD8 T cells based on cytokine secretion Eur J Immunol 29 4053 4059 573 Oelke M Moehrle U Chen JL Behringer D Cerundolo V Lindemann A Mackensen A 2000 Generation and purification of CD8 Melan A Specific Cytotoxic T Lymphocytes for Adoptive Transfer in Tumor Immunotherapy Clin Cancer Res 6 1997 2005 663 Oelke M Kurokawa T Hentrich I Behringer D Cerundolo V Lindemann A Mackensen A 2000 Functional Characterization of CD8 Antigen Specific Cytotoxic T Lymphocytes after Enrichment Based on Cytokine Secretion Compa rison with the MHC Tetramer Technology Scand J Immunol 52 544
3. Detection and enrichment of viable IFN y secreting leukocytes Detection and enrichment of IFN y secreting antigen specific T cells for enumeration and phenotypic analysis as well as for expansion and functional characterization 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 IFN y 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 Miltenyi Biotec 6 140 000 274 07 1 Description labeled with a second IFN y specific antibody the IFN y Detection Antibody conjugated to R phycoerythrin PE for sensitive detection by flow cytometry The IFN y 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 secreting cells The cells can now be used for cell culture or analysis Since viable cells are analyzed non specific background can be minimiz
4. 50 mL 25 cm 40 x 107 40 mL 250 mL 75 cm 80 x 107 80 mL 720 mL 162 cm 120 x 107 120 mL 900 mL 225 cm 140 000 274 07 7 Appendix 7 Appendix B Detection and enrichment of cytokine secreting cells from whole blood B1 Reagent and instrument requirements B2 Protocol B2 1 Antigen specific in vitro stimulation B2 2 Cytokine Secretion Assay B 2 3 Magnetic labeling B2 4 Magnetic separation The following special protocol can be used in combination with one of the Cytokine Secretion Assay Cell Enrichment and Detection Kits for human cells B1 Reagent and instrument requirements Cytokine Secretion Assay Kit for example IFN y Secretion Assay Cell Enrichment and Detection Kit PE 130 054 201 IL 2 Secretion Assay Cell Enrichment and Detection Kit PE 130 090 488 IL 4 Secretion Assay Cell Enrichment and Detection Kit PE 130 054 101 IL 10 Secretion Assay Cell Enrichment and Detection Kit PE 130 090 435 Miltenyi Biotec 28 140 000 274 07 7 Appendix Propidium iodide PI or 7 AAD to exclude dead cells from analysis MACS Columns and MACS Separators Column max number max number Separator of labeled cells of total cells MiniMACS OctoMACS with Column Adapter VarioMACS SuperMACS autoMACS 2x10 autoMACS A Note Column adapters are required to insert certain columns into VarioMACS Separator or SuperMACS Separator For details see MACS Separator d
5. cells in the enriched fraction x 100 x 100 abs of total CD4 cells before enrichment of IFN y CD4 cells in the analyzed sample of total CD4 cells in the analyzed sample Unstimulated control sample before enrichment after enrichment CD4 FITC CD4 FTIC anti IFN y PE anti IFN y PE 0 006 of the total CD4 T cell population secrete IFN y lt 1 IFN y CD4 T cells were enriched from 10 CD4 cells lt 0 0001 140 000 274 07 7 Appendix 7 Appendix A Flask and dish sizes for stimulation For in vitro stimulation see 4 2 step 2 the cells should be resuspended in culture medium containing 5 of human serum at 10 cells mL and 5x10 cells cm Both the dilution and the cell density are important to assure optimum stimulation The following table lists culture plate dish and flask sizes suitable for different cell numbers It also indicates the appropriate amount of medium to add total cell medium volume culture well number to add plate diameter 0 15 x 10 0 15 mL 96 well 0 64 cm 0 5 x 107 0 5 mL 48 well 1 13 cm 1 x 10 1 mL 24 well 1 6 cm 2 x 10 2mL 12 well 2 26 cm 5 x 10 5 mL 6 well 3 5 cm total cell medium volume culture dish number to add dish diameter 4 5 x 10 4 5 mL small 3 5 cm 10 x 10 10 mL medium 6cm 25 x 10 25 mL large 10 cm 50 x 10 50 mL extra large 15 cm total cell medium volume culture growth number to add flask area 12 x 107 12 mL
6. out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column directly onto the second column 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 Remove second column from separator place column on a suitable collection tube 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 Proceed to flow cytometric analysis see detailed protocol cell culture or other subsequent experiment A 140 000 274 07 7 Appendix Warnings Reagents contain sodium azide Under acidic conditions sodium azide yields hydrazoic acid which is extremely toxic Azide compounds should be diluted with running water before discarding These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop Warranty The products sold hereunder 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
7. should always be included in the experiment 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 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 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 of erythrocyte lysing solution room temperature Miltenyi Biotec 32 140 000 274 07 7 Appendix ee Labeling cells with Cytokine Catch Reagent f Resuspend cell pellet in 15 mL of cold buffer and transfer into a 15 mL conical propylene tube Centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely Resuspend pellet in 160 uL of cold medium Add 40 uL of Cytokine Catch Reagent mix well and incubate for 5 minutes on ice aA Cytokine secretion period Gy 1 Add7 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 Incubate cells in a closed tube for 45 minutes at 37 C under slow continuous rotatio
8. supplied with the column directly onto the second column MS 1 mL LS 5 mL Miltenyi Biotec 20 140 000 274 07 5 Detection and analysis of IFN y secreting T cells Place tube containing magnetically labeled cells in autoMACS Separator Choose separation program Posseld Collect the separated fractions from outlet port pos2 Proceed to analysis see section 5 cell culture or other subsequent experiment 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 IFN y secreting cells acquire all of the positive fraction For preparative purposes acquire an aliquot of the positive fraction to determine the performance of the cell enrichment To illustrate the analysis we describe the detection of IFN y secreting T cells using the IFN y Secretion Assay The detailed description including how to set gates should serve as a model for the analysis of your own sample Miltenyi Biotec 22 140 000 274 07 4 Protocol fo
9. 0 101 Miltenyi Biotec 16 140 000 274 07 4 Protocol for the IFN y Secretion Assay Incubate cells in closed tube for 45 minutes at 37 C under slow continuous rotation using the MACSmix tube rotator 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 77 Labeling cells with IFN y Detection Antibody 1 Put the tube on ice 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 Resuspend cell pellet in 80 uL of cold buffer per 10 total cells Add 20 uL of IFN y Detection Antibody PE per 10 total cells 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 Perc Po Mix well and incubate for 10 minutes on ice Wash cells by adding 10 mL of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C pipette off supernatant Miltenyi Biotec 18 140 000 274 07 4 Protocol for the IFN y Secretion Assay ae i Labeling cells with IFN y Catch Reagent rA 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 fewer than 107 cel
10. 549 970 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 Barr virus are primarily Th1 in function J Clin Invest 107 121 130 1035 7 Pittet MJ Zippelius A Speiser DE Assenmacher M Guillaume P Valmori D Lienard D Lejeune F Cerottini JC Romero P 2001 Ex vivo IFN y secretion by circulating CD8 T lymphocytes Implications of a novel approach for T cell monitoring in infectious malignant diseases J Immunol 166 7634 7640 1037 Becker C Pohla H Frankenberger F Sch ler T Assenmacher M Schendel DJ Blankenstein T 2001 Adoptive tumor therapy with T lymphocytes enriched through an IFN y capture assay Nature Medicine 7 10 1159 1162 1207 For further information visit our website www miltenyibiotec com Miltenyi Biotec 26 140 000 274 07 5 Detection and analysis of IFN y secreting T cells C Antigen specific CD4 T cells stained for secreted IFN y Sample stimulated with CMV lysate before enrichment after enrichment CD4 FITC CD4 FITC anti IFN y PE anti IFN y PE 0 257 of the total CD4 T cell population secrete IFN y see formula below The IFN y secreting CD4 T cells have been enriched to 70 8 1292 IFN y CD4 T cells were enriched from 10 CD4 cells 0 129 see formula below IFN y cells among CD4 IFN y cells among CD4 abs of IFN y CD4
11. CP 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 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 A lymphocyte gate based on forward and side scatter FSC SSC properties was activated prior to further gating to exclude monocytes and debris see A 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 Analysis of secreted IFN y PE versus CD4 FITC staining by viable lymphocytes is displayed see C 140 000 274 07 5 Detection and analysis of IFN y secreting T cells A Lymphocyte gate in the forward versus side scatter plot before enrichment after enrichment rol side scatter side scatter rool forward scatter forward scatter B Dead cell and monocyte exclusion in FL 2 versus FL 3 before enrichment after enrichment il
12. Cytokine Secretion Assays Miltenyi Biotec GmbH Friedrich Ebert Str 68 51429 Bergisch Gladbach Germany Phone 49 2204 83060 Fax 49 2204 85197 macs miltenyibiotec de Miltenyi Biotec Inc 2303 Lindbergh Street Auburn CA 95602 USA Phone 800 FOR MACS 1 530 888 8871 Fax 1 530 888 8925 macs miltenyibiotec com Miltenyi Biotec Pty Ltd Australia Phone 61 2 8877 7400 Fax 61 2 9889 5044 macs miltenyibiotec com au Miltenyi Biotec Shanghai Office Phone 86 21 62351005 Fax 8621 62350953 miltenyibiotec china com Miltenyi Biotec France Phone 33 1 56 98 16 16 Fax 33 15698 16 17 macs miltenyibiotec fr Miltenyi Biotec S r l Italy Phone 3951 6460 411 Fax 3951 64 60 499 macs miltenyibiotec it Miltenyi Biotec K K Japan Phone 81 3 56 46 8910 Fax 81 3 56468911 macs miltenyibiotec jp Miltenyi Biotec Asia Pacific Pte Ltd Singapore Phone 65 6238 8183 Fax 65 6238 0302 macs miltenyibiotec com sg Miltenyi Biotec S L Spain Phone 3491 512 12 90 Fax 34 91 512 12 91 macs miltenyibiotec es Miltenyi Biotec Ltd UK Phone 44 1483 799 800 Fax 44 1483 799 811 macs miltenyibiotec co uk 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 Z0 vZ7 000 0rL A This MAC
13. G1 conjugated to PE R phycoerythrin 1 mL Anti PE MicroBeads colloidal superparamagnetic MicroBeads conjugated to monoclonal mouse anti PE antibody mouse IgG1 For 50 tests with 10 cells All components are supplied as a suspension containing stabilizer and 0 05 sodium azide Store protected from light at 4 8 C Do not freeze The expiration dates are indicated on the vial labels A 1 Description Anti PE IFN y Catch Reagent IFN y Detection Antibody PE 1 1 Principle of the IFN y Secretion Assay Antigen specific T cells are analyzed and isolated using the IFN y 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 Subsequently an IFN y 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 IFN y binds to the IFN y Catch Reagent on the positive secreting cells These cells are subsequently Miltenyi Biotec 4 140 000 274 07 1 Description The MACS enrichment also enables further functional characteri zation 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
14. S product is for in vitro research use only and not for diagnostic or therapeutic procedures Cytokine Secretion Assays IFN y Secretion Assay Cell Enrichment and Detection Kit PE human For 50 tests with 107 cells Order No 130 054 201 Miltenyi Biotec 1 Description 1 Description Description 1 1 Principle of the IFN y Secretion Assay 1 2 Background and product applications 1 3 Reagent and instrument requirements Protocol overview Experimental set up 3 1 Controls 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 Protocol for the IFN y 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 Detection and analysis of IFN y secreting antigen specific T cells References Appendix A Flask and dish sizes for stimulation B Detection and enrichment of cytokine secreting cells from whole blood Miltenyi Biotec 2 140 000 274 07 Components Size Product format Storage 140 000 274 07 1 mL IFN y Catch Reagent anti IFN y monoclonal antibody mouse IgG1 conjugated to cell surface specific monoclonal antibody mouse IgG2a 1 mL IFN y Detection Antibody anti IFN y monoclonal antibody mouse Ig
15. ata sheets Optional Rotation device for tubes MACSmix tube rotator 130 090 753 Optional Pre Separation Filters 130 041 407 to remove cell clumps Miltenyi Biotec 30 140 000 274 07 Anticoagulant sodium heparin Buffer degassed Prepare a solution containing PBS phosphate buffered saline pH 7 2 0 5 BSA and 2 mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 with autoMACS Rinsing Solution 130 091 222 Keep buffer cold 4 8 C Culture medium e g RPMI 1640 130 091 440 containing 20 of human serum like autologous serum or AB serum do not use BSA or FCS because of non specific stimulation Erythrocyte lysing solution 1x prepare freshly from 10x stock solution 10x stock solution 41 4 g NH CI 1 55 M 5 g KHCO 100 mM 1 mL 0 5 M EDTA 1 mM adjust pH to 7 3 fill up to 500 mL with dd H O A Note Do not use FACS Lysing solution 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 PharMingen PE Cy5 Serotec ECD PC5 Coulter Immunotech etc they may also be recognized by the Anti PE MicroBeads A Note Upon activation of T cells TCR and some associated molecules like CD3 might be down regulated A Note For optimal sensitivity we recommend labeling of undesired non T cells such as monocytes with antibodies conjugated to PerCP e
16. ed by dead cell exclusion This provides highest sensitivity of analysis 1 2 Background and product applications The IFN y Secretion Assay Cell Enrichment and Detection Kit is designed for the detection isolation and analysis of viable IFN y 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 IFN y is induced IFN y is predominantly secreted by activated CD4 and CD8 memory and effector T cells and by NK cells upon activation 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 140 000 274 07 1 Description 1 3 Reagent and instrument requirements Buffer degassed Prepare a solution containing PBS phosphate buffered saline pH 7 2 0 5 BSA and 2 mM EDTA by diluting MACS BSA Stock Solution 130 091 376 1 20 with autoMACS Rinsing Solution 130 091 222 Keep buffer cold 4 8 C Culture medium e g RPMI 1640 130 091 440 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 for flow cytometric exclusion of dead cells For cell fixation and flow cytometric exclu
17. er and mix Centrifuge at 200xg for 10 15 minutes at 20 C Carefully remove supernatant Special protocols for whole blood You can start the IFN y 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 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 Miltenyi Biotec 14 140 000 274 07 3 Experimental set up 3 5 Combination with peptide MHC tetramer staining IFN y secreting cells can be analyzed simultaneously for peptide MHC tetramers by combining the IFN y Secretion Assay PE with APC conjugated peptide MHC tetramers For combination with PE conjugated peptide MHC tetramers the IFN y Secretion Assay Detection Kit APC 130 090 762 and the IFN y Secretion Assay Detection Kit FITC 130 090 433 are 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 3 6 Detection without prior enrichment Optional If the sample contains more than 0 01 0 1
18. g CD14 PerCP These cells can then be excluded together with PI stained dead cells by gating A 140 000 274 07 7 Appendix 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 A Do not use media containing any non human proteins like BSA or FCS because of non specific stimulation 8 oes Protocol for in vitro stimulation 1 Start with 5 mL of fresh sodium heparinized human blood containing about 10 lymphocytes ina50 mL conical polypropylene tube 140 000 274 07 7 Appendix 7 Appendix 2 Add the antigen or as a positive control 1 g 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 A negative control sample treated exactly the same as the antigen stimulated sample but without addition of antigen
19. ls use same volumes 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 Resuspend cell pellet in 80 uL of cold medium per 10 total cells Add 20 uL of IFN y Catch Reagent per 10 total cells mix well and incubate for 5 minutes on ice ah IFN y 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 IFN y secreting cells add per 10 total cells lt 5 10 cells mL 10 mL 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 5 140 000 274 07 4 Protocol for the IFN y Secretion Assay 4 4 Magnetic labeling 73 Magnetic labeling with Anti PE MicroBeads 1 Resuspend cell pellet in 80 uL of cold buffer per 10 total cells 2 Add 20 uL of Anti PE MicroBeads 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 Wash cells by adding 10 mL of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant Resuspend cell pellet in 500 uL of cold buffer For higher cell numbers than 5x10 use a dilution of 10 cells mL
20. ls first and take them into culture overnight but without adding the antigen see 4 2 step 2 Peptide is then added the next morning for 3 hours of stimulation directly followed by the IFN y Secretion Assay Proteins Upon stimulation with protein the cells can be analyzed for IFN y secretion 6 16 hours later It is possible to start the stimulation of the cells late in the afternoon and to perform the IFN y 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 IFN y secreting cells are stained with PE conjugated IFN y Detection Antibodies To identify cells of interest counterstaining for T cells with eg CD4 FITC 130 080 501 or CD8 FITC 130 080 601 is important 140 000 274 07 3 Experimental set up A Do not use tandem conjugates of phycoerythrin like Cy Chrome PharMingen PE Cy5 Serotec ECD PC5 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
21. mpletely 140 000 274 07 7 Appendix 77 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 Resuspend cell pellet in 160 uL of cold buffer Add 40 uL of Cytokine Detection Antibody PE Optional Add additional staining reagents e g 20 uL of CD4 FITC 130 080 501 or CD8 FITC 130 080 601 and CD14 PerCP Mix well and incubate for 10 minutes on ice 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 73 Magnetic labeling with Anti PE MicroBeads 1 Resuspend cell pellet in 160 uL of cold buffer 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 140 000 274 07 7 Appendix 7 Appendix Wash cells by adding 10 mL of cold buffer centrifuge at 300xg for 10 minutes at 4 8 C Pipette off supernatant completely Resuspend cell pellet in 500 uL of cold buffer Optional Take an aliquot for flow cytometric analysis and cell count of the fraction before enrichment 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 Colum
22. n using the MACSmix tube rotator 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 Miltenyi Biotec 34 140 000 274 07 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 Mix gently and incubate for 10 minutes at room temperature Rotate tube continuously using the MACSmix tube rotator 130 090 753 or turn tube several times during incubation Centrifuge cells at 300xg for 10 minutes at room temperature remove supernatant co
23. necessary to dilute the cells further during the cytokine secretion period and therefore a larger test tube will be needed 140 000 274 07 4 Protocol for the IFN y Secretion Assay see table below The dilution prevents non specific staining of cells not secreting IFN y 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 fewer 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 large amounts of dead cells they should be removed before starting the IFN y Secretion Assay e g by density gradient centrifugation or by using the Dead Cell Removal Kit 130 09
24. ns to achieve best results 1 Prepare two MS Columns per sample by rinsing with 500 uL cold buffer discard effluent Place first column into the magnetic field of a MACS Separator use column adapter with VarioMACS or SuperMACS Separator Optional Pass cells through Pre Separation Filters 130 041 407 to remove clumps Apply cell suspension onto the column Miltenyi Biotec 36 140 000 274 07 7 Appendix Magnetic separation using the autoMACS Separator ARefer to the auttoMACS User Manual for instructions on how to use the autoMACS Separator Prepare and prime autoMACS Separator Optional Pass cells through Pre Separation Filters 130 041 407 to remove clumps Place tube containing magnetically labeled cells in autoMACS Separator Choose separation program Posseld Collect the separated fractions from outlet port pos2 Proceed to flow cytometric analysis see detailed protocol cell culture or other subsequent experiment Miltenyi Biotec 38 140 000 274 07 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 Remove first column from separator place second column into the separator and put the first column on top of the second one Pipette 1 mL of cold buffer on top of the first column Immediately flush
25. of IFN y secreting cells you may be able to analyze IFN y secreting cells without prior enrichment see also IFN y Secretion Assay Detection Kit PE 130 054 202 The assay can also be performed directly starting from whole blood A detailed protocol is included in the data sheet of the IFN y Secretion Assay Detection Kit PE and is available from our website www miltenyibiotec com 140 000 274 07 4 Protocol for the IFN y Secretion Assay 8 oes Protocol for in vitro stimulation 1 Wash cells by adding medium centrifuge at 300xg for 10 minutes 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 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 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 4 3 Cytokine Secretion Assay General considerations A The assay is optimized for cell samples containing lt 5 of total IFN y secreting cells If gt 5 of IFN y secreting cells are expected it is
26. of a product for a particular purpose There are no warranties expressed or implied which extend beyond the technical specifications of the products Miltenyi Biotec GmbH s 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 MACS is a registered trademark of Miltenyi Biotec GmbH 140 000 274 07
27. 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 IFN y secreting cells do not allow conclusions on the performance e g sensitivity of the IFN y Secretion Assay Miltenyi Biotec 10 140 000 274 07 2 Protocol overview 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 3 16 hours 37 C 4 C IFN y Secretion Assay see 4 3 e Labeling with IFN y Catch Reagent 5 minutes on ice e IFN y secretion period 45 minutes 37 C e Labeling with IFN y 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 over 2 MS or LS Columns or with the autoM ACS E Detection analysis see 5 cell culture or subsequent experiment 140 000 274 07 3 Experimental set up 3 2 Kinetics of restimulation and proposed time schedule Peptides Upon stimulation with peptide the cells can be analyzed for IFN y secretion 3 6 hours later It is possible to prepare the cel
28. r the IFN y Secretion Assay 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 Remove the second column from separator place the column on a suitable collection tube 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 by flow cytometry the medium should not contain phenol red Proceed to analysis see section 5 cell culture or other subsequent experiment Magnetic separation using the autoMACS Separator A Refer to the autoMACS User Manual for instructions on how to use the autoMACS Separator 1 Prepare and prime autoMACS Separator 2 Optional Pass cells through Pre Separation Filters 130 041 407 to remove clumps A 140 000 274 07 5 Detection and analysis of IFN y secreting T cells 10 human PBMC of a CMV donor have been restimulated for 16 hours with and without CMV lysate 5 ug mL Biowhittaker The IFN y Secretion Assay was performed on the stimulated and the unstimulated sample Counterstaining of T cells was performed using CD4 FITC Monocytes were stained with CD14 Per
29. sion of dead cells the Fixation and Dead Cell Discrimination Kit 130 091 163 is recommended Optional Staining reagents such as CD4 FITC 130 080 501 or CD8 FITC 130 080 601 and CD14 PerCP 140 000 274 07 1 Description MACS Columns and MACS Separators Column max number max number Separator of labeled cells of total cells MiniMACS OctoMACS with Column Adapter VarioMACS Super MACS MidiMACS with Column Adapter VarioMACS SuperMACS autoMACS 2x 108 autoMACS A Note Column adapters are required to insert certain columns into VarioMACS Separator or SuperMACS Separator For details see MACS Separator data sheets Refrigerated centrifuge 4 8 C Rotation device for tubes MACSmix tube rotator 130 090 753 Optional Pre Separation Filters 130 041 407 to remove cell clumps Miltenyi Biotec 8 140 000 274 07 3 Experimental set up 3 Experimental set up Negative control For accurate detection of IFN y secreting antigen specific cells a negative control sample should always be included This will provide information about IFN y 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
30. staining and increase sensitivity 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 IFN y secreting cells can be analyzed simultaneously for IL 2 or IL 10 production by two color cytokine analysis combining the IFN y Secretion Assay with the IL 2 Secretion Assay Detection Kit APC 130 090 763 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 Miltenyi Biotec 12 140 000 274 07 4 Protocol for the IFN y Secretion Assay 4 Protocol for the IFN y 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 buff
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