HLA-B27 HLA-B27 CaliBRITE Beads FACSComp HLA-B27 Calibration Beads HLA-B27 HLA-B27
1. HLA-B27 1.1 HLA-B27 HLA Major Histocompatibity Complex MHC HLA HLA-A -B -C HLA HLA-D/DR -DP -DQ B HLA HLA HLA HLA-B27 MHC HLA-B27 90% HLA-B27 5-10% HLA-B27 HLA-B27 Reiter s HLA-B27 70-90% HLA-B27 50-60% HLA-B27 40-50% HLA-B27 5-10% HLA-B27 1.2 HLA-B27 1.2.1 HLA Terasaki McClelland HLA 100% Becton Dickinson Immunocytometry System - 1 -
1.2.2 HLA-B27 100% 97.4% 1.2.3 HLA-B27 BD HLA-B27 HLA-B27 T- HLA-B27 Reagent A Anti-HLA-B27 FITC/CD3 PE 1.5ml FITC -HLA-B27 GS145.2 HLA-B27 PE -CD3 SK7 T Reagent B 10X Lysing Solution 30ml 10 Reagent C Calibration beads for FL1 1.5ml CaliBRITE beads HLA-B27 calibration beads Cutoff marker HLA-B27 FSC-FL2 CD3 CD3+T FSC FSC 50% CD3+T T HLA-B27 GS145.2 HLA-B27 HLA-B7 HLA -B T HLA T -HAL-B27 FITC HLA-B7 HLA-B27 HLA-B27 FSC/FL2 HLA-B27 FITC Cutoff marker HLA-B27 HLA-B27 Cutoff marker Becton Dickinson Immunocytometry System - 2 -
2. K 3 EDTA heparin ACD 1ml ACD Heparin 24 2.1 ( ) 1) 1X FACS Lysing PBS with 1% paraformaldehyde 1X FACS Lysing 2) 3) 30ul A 4) 50ul 3 15-20 4 2ml 1X 10-12 12 5 5 300xg 6 50ul 7 2ml 0.1% Sodium Azide PBS 5 200xg 8 50ul 9 0.5ml 1% paraformaldehyde 30 10 2-8 24 Becton Dickinson Immunocytometry System - 3 -
3. FACS Calibur HLA-B27 HLA-B27 Cutoff marker FL1 HLA-B27 CaliBration beads 3.1 HLA-B27 1 FACSComp CaliBRITE beads Lyse/Wash HLA-B27 2 HLA-B27 Calibration Beads FSC FL1 PMT HLA-B27 3 HLA-B27 3.2 Calibrite beads 4 C (usually prepared every 3 days, more frequently when necessary). FACSFlow (BD Products, 342003) CaliBRITE beads 1-2 ml FACSFlow BLANK FITC PE HLA-B27 Unlabled (1 ml FACSFlow) x Mixed Beads (2 ml FACSFlow) x x x HLA-B27 (1 ml FACSFlow) x Becton Dickinson Immunocytometry System - 4 -
3.3 FACSComp Sign In Set Up PMT Comp Sens HLA-B27 Summary HLA-B27 3.4 FACSComp 2 FACSComp 2 FACSComp PMTs FACScomp FACSComp Sign In Accept Becton Dickinson Immunocytometry System - 5 -
Set Up 1. : Lyse/Wash 2. CaliBRITE FITC- / PE- / Unlabeled- beads. 3. Run PMT unlabeled bead Start PMTs Set Successfully Next Comp mixed beads Start : FL1 - % FL2, FL2 - % FL1 and/or FL3 - % FL2 Compensation Set Successfully Next Sens mixed bead Start Summary Summary Report Quit File menu Quit OK Becton Dickinson Immunocytometry System - 6 -
Becton Dickinson Immunocytometry System - 7 -
3.4 FACSComp HLA-B27 FACSComp HLA-B27 FACScomp FACSComp Sign In Accept Set Up HLA-B27 Calib Beads Lot Number Suffix HLA-B27 Lot Number Suffix Run Beads Suffix HLA-B27 Suffix FL1 PMT Cutoff marker HLA-B27 B27 Calibration Bead Start FSC FL1 PMT Summery Calibration Report FSC FL1 HLA-B27 Samples Becton Dickinson Immunocytometry System - 8 -
4. HLA-B27 HLA-B27 T HLA-B27 T HLA-B27 Calibration Bead Marker 4.2 HLA-B27 Sign In Set Up FACSComp Samples Acquisition Analysis Lab Report Summary FACSComp HLA-B27 Marker Becton Dickinson Immunocytometry System - 9 -
4.3 HLA-B27 CaliBRITE Beads FACSComp PMT Cytometer Instrument Settings BD File:/Instrument Settings File:/Calib B27 4.4 HLA-B27 1 Sign In 2 Set Up Data sources From Cytometer 15000-50000 15000 HLA-B27 Case Number Location Until Next Button Pressed 3 FACSComp FACSComp HLA-B27 Skip FACSComp Becton Dickinson Immunocytometry System - 10 -
4 Samples Case Number Run Test Instrument Settings Cytometer Instrument Settings BD File:/Instrument Settings File:/Calib B27 Becton Dickinson Immunocytometry System - 11 -
5 Acquisition HI HLA-B27 Acquire Pause Stop Samples Restart Acquire Skip Resume Analyze 15000 6 Analysis FCS Sign In Set Up Samples FL1 FL1 HLA-B27 HLA-B27 FSC-SSC FSC-FL2 FL1 FL1 FL1 HLA-B27 Becton Dickinson Immunocytometry System - 12 -
Next FCS Sign In Set Up Samples FL1 FL1 HLA-B27 HLA-B27 More Tests Samples 7 File Quit Schedule document Becton Dickinson Immunocytometry System - 13 -
4.5 2% 1. 2. 3. 4. CD3+ CD3-1. 2. (24 ) 3. 4. 1. 2. 3. Becton Dickinson Immunocytometry System - 14 -
6. HLA-B27 HLA-B27 1 HLA-B27 Sign In 2 Accept Set Up From Data Files Set Up 3 Accept FACSComp Samples 4 Add Samples Add Done Samples 5 Run Test 6 Next 7 Continue Continue 8 Summary Report Quit File menu Quit Becton Dickinson Immunocytometry System - 15 -
7. 7.1 BD FACSComp CaliBRITE beads HLA-B27 calibration beads BD HLA-B27 HLA-B27 HLA-B27 1 2% T 2 CD3+ CD3-7.2 BD FACSCalibur FACSort FACScan FACStation HLA-B27 Macintosh 7.1 FACSComp 7.3 HLA-B27 FCS Data file HLA-B27 FCS2.0 Schedule Document File.sch Becton Dickinson Immunocytometry System - 16 -
Laboratory Report File PICT FL1 FSC-SSC FSC-FL2.lab Summary Report File TEXT Schedule document Summary report.sum 7.4 7.4.1 Apple About HLA-B27 HLA-B27 About HLA-B27 HLA-B27 7.4.2 File New Schedule HLA-B27 schedule document schedule Open Schedule schedule document Open Lab Report Lab Report Close schedule document Lab Report Save schedule document Save As schedule document Save Report Lab Report Summary Report Page Setup Print Quit Becton Dickinson Immunocytometry System - 17 -
7.4.3 Edit Undo Cut document Copy Paste Clear Select All Show Clipboard 7.4.4 Cytometer Detector/Amps PMT Threshold Compensation Status Instrument Setting 7.4.5 Preference Schedule 1 HLA-B27 Preference 2 Export Tab( Macintosh, Comma, ). 3 Printing Never 4 Save After Each Entry Becton Dickinson Immunocytometry System - 18 -