Created by judith.eckl Page 1 of 8 09/06/2011 SOPVII-7 Panel X: NK-characterization Date: Author: Petra Prinz, Judith Eckl Experimenter: Date: 08/06/2011 Experiment description: Version: 1.0 Start: End: Background information: Human NK cells are the major effector cells of the innate immunity and play a important role in the interplay of innate and adaptive immunity. Human NK cells are characterized as CD3 - CD56 + cells. Different subtypes can be distinguished according to the expression level of CD56 and CD16. NKp46, NKp30, CD57, CD94, Perforin, Granzym B and NKG2D are markers that can correlate with the effector function of the NK-cells
Created by judith.eckl Page 2 of 8 09/06/2011 Material: Live/Dead Fixable Dead Cell Stain Kit [Invitrogen: Cat. No: L23105] ArC Amine Reactive [Invitrogen: Cat. No: A10346] PBS w/o Mg Ca [Invitrogen: Cat. No: 14190094] BSA [Sigma Cat. No: A7906-100G] EDTA [Invitrogen Cat. No: AM9260G] NaN 3 [Sigma Cat. No: 08591-1ML-F] Foxp3 Staining Buffer Set (ebioscience, Cat. No: 00-5523) Antibodies [see list page 2] Aqua bidest. Compensation beads Anti-mouse Ig, κ [ Cat. No: 552843 ] Compensation beads Anti-rat/hamster Ig, κ [ Cat. No: 552844 ] U-bottom 96well plates [ Falcon; Cat. No: FalC353910] 15ml Tube [ Falcon; Cat. No: 5000741] 50ml Tube [ Falcon; Cat. No: 5000743] Serologic Pipettes 5ml [Greiner Bio-One; Cat. No: 606180] Serologic Pipettes 10ml [Greiner Bio-One; Cat. No: 607180] Serologic Pipettes 25ml [Greiner Bio-One; Cat. No: 760180] Pipette tips 0.5-10µl [Peske; Cat. No: 212100] Pipette tips 1-200µl [Corning; Cat. No: CORN4783] Pipette tips 100-1000µl [Greiner Bio-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 Flowcytometer (e.g. LSR II ())
Created by judith.eckl Page 3 of 8 09/06/2011 Antibodies/Cellstains: 1 Marker Fluorescence Antibody cellular isotyp localization dilution company Cat. No CD3 Pacific surface 1:20 558117 CD56 APC surface 1:20 Beckmann/C oulter IM2474 CD16 Alexa 700 surface 1:25 Caltag MHCD16 29 NKp46 PE surface 1:5 557991 Perforin FITC intracellular 1:10 558574 live/dead UV 1:200 Invitrogen L-23105 2 Marker Fluorescence Antibody cellular isotyp localization dilution company Cat. No CD3 Pacific surface 1:20 558117 CD56 APC surface 1:20 Beckmann/C oulter IM2474 CD16 Alexa 700 surface 1:25 Caltag MHCD16 29 Granzym MCA212 PE intracellular 1::50 Serotec e B 0 CD69 FITC surface 1:10 560969 live/dead UV 1:200 Invitrogen L-23105 3 Marker Fluorescence Antibody cellular isotyp localization dilution company Cat. No CD3 Pacific surface 1:20 558117 CD56 PE surface 1:10 Pharmigen 555516 CD16 Alexa 700 surface 1:25 Caltag MHCD16 29 NKG2D APC surface 1:20 Pharmigen 558071 CD94 FITC surface 1:10 Pharmigen 555888 live/dead UV 1:200 Invitrogen L-23105
Created by judith.eckl Page 4 of 8 09/06/2011 4 Marker Fluorescence Antibody cellular isotyp localization dilution company Cat. No CD3 Pacific surface 1:20 558117 CD56 APC surface 1:20 Beckmann/C oulter IM2474 CD16 Alexa 700 surface 1:25 Caltag MHCD16 29 NKp44 PE surface 1:5 558563 CD57 FITC surface 1:10 ebioscience 11-0577- 41 live/dead UV 1:200 Invitrogen L-23105 Flowcytometer Laser configuration Fluorescence -Parameter Laser Detector Filter FSC PE-Cy7 PerCP-Cy5.5 PE FITC SSC APC-Alexa780 APC Horizon V500 PB/Pacific live/dead fixable blue Red Red Violet Violet UV FSC A B E F G A C A B B 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 5 of 8 09/06/2011 Protocoll FACS-staining Reagent set-up: 1. Before starting with the experiment make sure that you have read and understood the whole protocol and that all necessary materials are available 2. Dye preparation Live/Dead Fixable Dead Cell Stain Kit. Bring one vial of the fluorescent reactive dye (Component A) and the vial of anhydrous 3. DMSO (Component B) to room temperature before removing the caps.. Add 50 μl of DMSO (compound B) to the vial of reactive dye (compound A). 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. 4. Preparation of Fixation/Permeabilisation working solution: 1 Part Fixation/Permeabilisation Concentrate is mixed with 3 parts Fixation/Permeabilisation Diluent of the Foxp3 Staining Buffer Set. Per well of a 96well Plate 250µl of working solution has to be prepared. Buffer has to be prepared freshly on the day of experiment! 5. Preparation of the Permeabilisation buffer: The 10x Permeabilisation buffer is diluted with aqua bidest to 1x concentration 6. Preparation of FACS-Buffer: 200ml PBS w/o Mg&Ca 2%BSA, 2mM EDTA and 0,002% NaN 3 are mixed 7. 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. 8. To minimize the risk mixing up the samples note all samples and their location on the plate layout on page 5. 9. 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. Air bubbles should be avoided as much as possible.
Created by judith.eckl Page 6 of 8 09/06/2011 Staining compensationsbeads live/dead fixable blue stain 1. Gently vortex the ArC Amine beads; beads should be at RT! 2. Add 1 drop reactive (green cap) in a well 3. Add 1µl Live/Dead Fixable -solution. Mix well! 4. Incubate 30 Minutes at 4 C in the Dark 5. Wash the with PBS, centrifuge 1600 rpm 6 Minutes 4 C 6. Resuspend the beads in 100 µl FACS-Buffer 7. Add 1 drop negative- (white cap). Mix well! 8. Acquire the beads together with the samples at the flow cytometer. Staining compensationsbeads antibody staining 1. Gently vortex the compensation beads; beads should be at RT! 2. Add 15µl of positive and negative beads to each single well and add additionally 75µl of PBS. 3. Add 1µl of the specific antibody according to the plate layout on page 5.Mix well! 4. Incubate 30 Minutes at 4 C in the Dark 5. Wash the with PBS, centrifuge 1600 rpm 6 Minutes 4 C 6. Resuspend the beads in 100 µl FACS-Buffer 7. Acquire the beads together with the samples at the flow cytometer.
Created by judith.eckl Page 7 of 8 09/06/2011 Plate layout Staining NK-characterization 1 2 3 4 5 6 7 8 9 10 11 12 A PE-Cy7 APC-Alexa 780 V500 FITC PerCP-Cy5.5 PE APC PB Alexa 700 UV B C D E F G H
Created by judith.eckl Page 8 of 8 09/06/2011 Antibody-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. Avoid cross contamination and blot the plate on a clean tissue. 4. Per sample 0, 5µl Live/Dead Fixable Solution is mixed with 100µl PBS. This is sufficient for 0, 5*10 5 to 1*10 6 Cells. Always prepare for 2 samples more than necessary. 5. Loosen the pellet with gentle vortexing and resuspend the cells in the 100µl Fixable blue/pbs 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, label the tube and spin down the solution. Always prepare for 2 samples more than necessary. 8. Wash the cells with 200µl PBS, 9. centrifugation 1600rpm 6 Minutes 4 C, discard the supernatant 10. Loosen the pellet with gentle vortexing and resuspend the cells in 50µl FACS buffer and add the appropriate amount of surface antibody working solution. Mix well! 11. Incubate the cells for 30 Minutes at 4 C in the Dark. Prepare the antibody compensation beads according to the instructions on page 4. 12. Wash the cells with 200µl FACS-Buffer 13. centrifugation 1600rpm 6 Minutes 4 C, discard the supernatant 14. Loosen the pellet with gentle vortexing and at 200µl of the freshly prepared Fixation/Permeabilisation working solution to all samples and mix well 15. Incubate for 30 Minutes at 4 C in the Dark. 16. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 17. wash the cells with 200µl Permeabilisation Buffer, 18. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 19. Loosen the pellet with gentle vortexing and resuspend the cells in 50µl Permeabilisation buffer and add the intracellular antibodies 20. Incubate for 30 Minutes at 4 C in the dark 21. Wash the cells with 200µl Permeabilisation Buffer 22. centrifugation 1800rpm 6 Minutes 4 C, discard the supernatant 23. repeat step 21 24. Loosen the pellet with gentle vortexing and resuspend the cells in 100µl FACS-Buffer. Optional: Resuspend cells in 100µl 1%PFA in PBS and store sealed plate in the fridge overnight, if cells can not be measured the same day. 25. Measure and acquire the data at the Flow cytometer (e.g. LSR II, ). Be aware that permeabilized cells are smaller as the non-permeabilzed cells and adjust the FSC/SSC settings accordingly.