Created by judith.eckl Page 1 of 6 09/06/2011 SOPVII-7 Panel VII: Treg-FCS-staining Date: uthor: Judith Eckl Experimenter: Date: 08/05/2011 Experiment description: Version: 1.0 Start: End: ackground information: Natural human regulatory T-cells are according to the current knowledge characterized as CD3 + CD4 + CD25 ++ CD127 +- CD45R - FoxP3 + cells. These cells can be isolated from the peripheral blood and show suppressing effects on the proliferation of effector T-cells. In this staining Panel the marker CD39 is included as it is published as a marker to distinguish naïve regulatory T-cells from memory regulatory T-cells. Material: Live/Dead Fixable Dead Cell Stain Kit [Invitrogen: Cat. No: L23105] rc mine Reactive [Invitrogen: Cat. No: 10346] PS w/o Mg Ca [Invitrogen: Cat. No: 14190094] S [Sigma Cat. No: 7906-100G] EDT [Invitrogen Cat. No: M9260G] NaN 3 [Sigma Cat. No: 08591-1ML-F] Foxp3 Staining uffer Set (eioscience, Cat. No: 00-5523) ntibodies [see list page 2] qua bidest. Compensation beads nti-mouse Ig, κ [ D Cat. No: 552843 ] Compensation beads nti-rat/hamster Ig, κ [ D Cat. No: 552844 ] U-bottom 96well plates [D Falcon; Cat. No: FalC353910] 15ml Tube [D Falcon; Cat. No: 5000741] 50ml Tube [D Falcon; Cat. No: 5000743] Serologic Pipettes 5ml [Greiner io-one; Cat. No: 606180] Serologic Pipettes 10ml [Greiner io-one; Cat. No: 607180] Serologic Pipettes 25ml [Greiner io-one; Cat. No: 760180] Pipette tips 0.5-10µl [Peske; Cat. No: 212100]
Created by judith.eckl Page 2 of 6 09/06/2011 Pipette tips 1-200µl [Corning; Cat. No: CORN4783] Pipette tips 100-1000µl [Greiner io-one; Cat. No: 741045] Reagent Reservoir [Costar; Cat. No: 4870] Equipment: Centrifuge with cooling function Ice Vortexer Table centrifuge calibrated single channel pipette in the range of 0,5 1000µl calibrated 8 or 12-channel channel pipette in the range of 20-300µl fridge Flow cytometer (e.g. LSR II (D)) ntibodies/cellstains: Marker ntibody- Isotyp Fluorescenz Lab-Id Company Cat. No ntibody per sample CD3 migg1 P 209 D 557832 5µl CD4 migg1 PC-lexa 780 206 eioscience 47-0049-42 3µl CD25 migg1 FITC 19/20 D 555431 3µl CD45R migg2b PerCP-Cy5.5 240 eioscience 45-0458-73 5µl CD127 migg1 PE 244 D 130-094-889 5µl CD39 migg1 PE-Cy7 254 eioscience 25-0399-71 5µl FoxP3 FoxP3 Isotyp Rat IgG2a, κ Rat IgG2a, κ PC 45 eioscience 17-4776-73 5µl PC 7 eioscience 12-4301-81 5µl live/dead UV Invitrogen L-23105 0,5 µl ntibody per samples Flow cytometer Laser configuration Fluorescence -Parameter Laser Detector Filter FSC PE-Cy7 PerCP-Cy5.5 PE FITC SSC PC-lexa780 PC Horizon V500 P/Pacific live/dead fixable blue Red Red Violet Violet UV FSC E F G C 780/60 695/40 695/40 575/26 530/30 488/10 780/60 660/20 525/50 450/50 450/50 755LP 685LP 550LP 505LP 755LP 505LP
Created by judith.eckl Page 3 of 6 09/06/2011 Protocoll FCS-staining Reagent set-up: 1. efore starting with the experiment make sure that you have read and understood the whole protocol and that all necessary materials are available 2. For every sample a FMO Control that contains the Isotyp for the FoxP3 antibody has to be included to allow proper setting of the gate in Data analysis. 3. Dye preparation Live/Dead Fixable Dead Cell Stain Kit. ring one vial of the fluorescent reactive dye (Component ) and the vial of anhydrous 4. DMSO (Component ) to room temperature before removing the caps.. dd 50 μl of DMSO (compound ) to the vial of reactive dye (compound ). Mix well and visually confirm that all of the dye has dissolved. One vial can be used for 80 stainings. The remaining Live/Dead Fixable -solution is stable for 8 weeks at -20 C. 5. Preparation of Fixation/Permeabilisation working solution: 1 Part Fixation/Permeabilisation Concentrate is mixed with 3 parts Fixation/Permeabilisation Diluent of the Foxp3 Staining uffer Set. Per well of a 96well Plate 250µl of working solution has to be prepared. uffer has to be prepared freshly on the day of experiment! 6. Preparation of the Permeabilisation buffer: The 10x Permeabilisation buffer is diluted with aqua bidest to 1x concentration 7. Preparation of FCS-uffer: 200ml PS w/o Mg&Ca 2%S, 2mM EDT and 0,002% NaN 3 are mixed 8. The staining is performed in a 96roundbottom plate. The plate is signed with the date of the experiment and the name of the person performing the experiment. Make sure to cover the plate during all incubation phases and centrifugation steps to minimize contaminations. Make sure that a pellet is visible after centrifugation. 9. To minimize the risk mixing up the samples note all samples and their location on the plate layout on page 5. 10. To wash the wells the washing solution is filled into a clean reagent reservoir and the solution is pipetted with a multichannel pipette into the wells. The cells are washed with careful up and down pipetting. ir bubbles should be avoided as much as possible.
Created by judith.eckl Page 4 of 6 09/06/2011 Staining compensation beads live/dead fixable blue stain 1. Gently vortex the rc mine beads; beads should be at RT! 2. dd 1 drop reactive (green cap) in a well 3. dd 1µl Live/Dead Fixable -solution. Mix well! 4. Incubate 30 Minutes at 4 C in the Dark 5. Wash the with PS, centrifuge 1600 rpm 6 Minutes 4 C 6. Resuspend the beads in 100 µl FCS-uffer 7. dd 1 drop negative- (white cap). Mix well! 8. cquire the beads together with the samples at the flowcytometer. Staining compensation beads antibody staining 1. Gently vortex the compensation beads; beads should be at RT! 2. Please note that the FoxP3 antibody is a rat antibody and needs the appropriate die nti-rat/hamster Ig, κ Compensation beads as well as the mouse antibodies need the nti-mouse Ig, κ Compensation beads. 3. dd 15µl of positive and negative beads to each single well and add additionally 75µl of PS. 4. dd 1µl of the specific antibody according to the plate layout on page 5.Mix well! 5. Incubate 30 Minutes at 4 C in the Dark 6. Wash the with PS, centrifuge 1600 rpm 6 Minutes 4 C 7. Resuspend the beads in 100 µl FCS-uffer 8. cquire the beads together with the samples at the flow cytometer.
Created by judith.eckl Page 5 of 6 09/06/2011 Plate layout Staining Panel VII 1 2 3 4 5 6 7 8 9 10 11 12 PE-Cy7 PC-lexa 780 V500 FITC PerCP-Cy5.5 PE PC beads P UV C D E F G H
Created by judith.eckl Page 6 of 6 09/06/2011 FoxP3 staining: 1. 1*10 6 cells per well are given into the 96well plate according to the plate layout (page 5). Don t forget the FMO Control! 2. centrifugation 1600rpm 6 Minutes 4 C 3. Discard the supernatant. void cross contamination and blot the plate on a clean tissue. 4. Per sample 0, 5µl Live/Dead Fixable Solution is mixed with 100µl PS. This is sufficient for 0, 5*10 5 to 1*10 6 Cells. lways prepare for 2 samples more than necessary. 5. Loosen the pellet with gentle vortexing and resuspend the cells in the 100µl Fixable blue/ps solution as prepared in step 4 and mix well. Prepare the live/dead compensation beads according to the instructions on page 4. 6. Incubate the cells for 30 Minutes at 4 C in the Dark. 7. In the meantime prepare an antibody working solution containing all surface antibodies, lable the tube and spin down the solution. lways prepare for 2 samples more than necessary. 8. Wash the cells with 200µl PS, 9. centrifugation 1600rpm 6 Minutes 4 C, discard the supernatant 10. Loosen the pellet with gentle vortexing and resuspend the cells in 50µl FCS buffer and the appropriate amount of surface antibody working solution, mix well and incubate the cells for 30Min at 4 C in the Dark. Prepare the antibody compensation beads according to the instructions on page 4. 11. Wash the cells with 200µl FCS-uffer 12. centrifugation 1600rpm 6 Minutes 4 C, discard the supernatant 13. Loosen the pellet with gentle vortexing and at 200µl of the freshly prepared Fixation/Permeabilisation working solution to all samples and mix well 14. Incubate for 60 Minutes at 4 C in the Dark. 15. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 16. wash the cells with 200µl Permeabilisation uffer, 17. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 18. Loosen the pellet with gentle vortexing and resuspend the cells in 50µl Permeabilisation buffer and add the FoxP3 antibody to the FoxP3 samples and respectively the isotype antibody to the FMO-controls, 19. Incubate for 60 Minutes at 4 C in the dark 20. Wash the cells with 200µl Permeabilisation uffer 21. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 22. repeat step 21 23. Loosen the pellet with gentle vortexing and resuspend the cells in 100µl FCS-uffer. FoxP3 stained cells have to be analyzed the same day sine they loose their FoxP3 signal when stored overnight even if they are fixed with PF. 24. Measure and acquire the data at the Flow cytometer (e.g. LSR II, D). e aware that permeabilized cells are smaller as the non-permeabilized cells and adjust the FSC/SSC settings accordingly.