FACSCalibur. FACS200 Handbook Pro DNA. FACSCalibur FACSCalibur. FACS Calibur. FSC Diode 488 nm SSC PMT 488 nm. FACS200 Handbook Pro 1
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1 FACSCalibur FACS200 Handbook Pro DNA FACSCalibur FACSCalibur 2. FACS Calibur 488 nm FACS Calibur FSC Diode 488 nm SSC PMT 488 nm FL1 PMT nm FL2 PMT nm FL3 PMT >650 nm FACS200 Handbook Pro 1
2 3. LO 12 µl /min MED 35 µl /min HI 60 µl /min RUN STANDBY PRIME PRIME STANDBY 4. Sheath Filter Air filter VENT TOGGLE FACS200 Handbook Pro 2
3 um 5. Sample Injection Tube Tube Support Arm Droplet Containment System FACS200 Handbook Pro 3
4 1.2 Macintosh X 10 5 cells/ml 0.5 ml 2. FALCON FALCON (Cat. No.2235) µm 4. DNA CV DNA DNA Content Analysis of Fixed Cells With Propidium Iodide DNA Content Analysis of Cell Lines Utilizing Trypsin DNA Content Analysis of Tissue Samples DNA Paraffin Embedded Tissue Samples Hypoploidy Analysis Hypoploidy Analysis daisy_kuo@bd.com Kenny_liu@bd.com FACS200 Handbook Pro 4
5 2.1 FACS Calibur MO FACS Flow ( 4L) ml ( 4L) 6. Pressurize PRIME 9. 1ml PBS HIGH RUN FACS Calibur 1. 2 ml FACSClean 10%Bleach 1 ml 2. HI RUN FACSClean Standby PRIME 4. 2 ml FACSRinse (2% Triton X-100) ml dh2o ml dh2o 7. STANDBY ml 9. VENT 10. File Quit ( Don t save ) BD 11. Special Shutdown FACS200 Handbook Pro 5
6 3.1 Calibur 1. * 1 connect to cytometer 2. VENT * 2 VENT RUN 3.2 CellQuest Pro 3. CELLQuest Pro Untitled 4. Acquire Connect to Cytometer Browser FACS200 Handbook Pro 6
7 5. Inspector Dot Plot FACS200 Handbook Pro 7
8 6. Edit Duplicate Edit Select All Inspector 3 Objects Plot Type Acquisition 9. P1 P2 X Y FSC-H 1024 SSC-H 1024 X Y FL1-W 1024 FL1-A 1024 FL1-A 1024 FL2-H PI FL2-A DNA FL2-W FSC SSC FACS200 Handbook Pro 8
9 3.2 Instrument settings Detector/ Amps Threshold Compensation Detector/Amps -- Threshold -- Compensation 10. Detectors/Amps DDM FL2 FL1DDM DNA FL2-W FL2-A 11. Cytometer Detectors/Amps Detectors/Amps Detectors/Amps FSC SSC FL2 LIN 12. Cytometer Threshold Threshold Threshold FL2-H 24 DNA (Ghost Cells) 13. LO RUN Acquisition Control Setup Acquire FACS200 Handbook Pro 9
10 FL2-H 14. FL2-A FL2-H ( 400 ) G0/G1 FL2-A FL2-W /FL2-A FL2-W Amp Gain 3.00 G0/G1 FL2-W FL2-A FL2-W DNA G1 DNA FL2-A G2 G2 DNA b G2 G1 G2 a FL2-W G2 DNA c Singlet Gating 16. FL2-W/FL2-A Inspector Multicolor Gating FACS200 Handbook Pro 10
11 17. Region FL2-W/FL2-A R1 Region R1 R1 Gates Region list R1 Delete R1 R1 18. FL2-A Inspector No Gate G1=R1 FSC/SSC Scatter Plot 19. FSC/SSC FSC (Voltage) E00 Amp Gain FSC E-1 FSC E01 SSC 20. Acquisition Control Pause Abort 21. Standby DNA Browser Directory Change Your Folder Select Your Folder FACS200 Handbook Pro 11
12 23. Browser File Change 24. Parameter Settings P1-P5 P1 Size, P2 Granularity, P4 PI, 25. Browser Global Settings Acquisition & Storage Settings Inspector Collection Criteria All Return Counters 26. Acquire Counters 27. HIGH RUN Acquisition Control Setup Setup CellQuest Pro Your Folder: Acquire CellQuest Pro FACS200 Handbook Pro 12
13 28. Acquire CD-RW Flash Drive PC Trash File Save Document As 31. Cytometer Instrument Setting Instrument Setting print 32. Save SAVE Your Folder /Your Setting1 Save Done 33. Acquire Disconnect to Cytometer 34. File Quit ( Don t save ) ml 5 1 ml HI RUN 2 ml DI water 1 ml DI water STANDBY FACS200 Handbook Pro 13
14 FACS200 Handbook Pro 14
15 ModFit LT Verity Software House DNA G1/S/G2+M DNA (DNA Index) 65 DNA, ModiFit LT : (1) Automatic Analysis (2) Manual Analysis (3) Synchronization (4) Proliferation ModiFit LT ModFit LT OK S-phase 2. Edit menu S-Phase Cutoffs Activate Assessment System S Phase 3. OK ModFit LT S S Phase ListMode Data 4. File Open File Demodata.001 Mac HD:/Demo Folder:/ Demodata.001 Open 5. Choose Parameter For Analysis FL2-A DNA OK 6. Define Gate 1 Define Gate Parameter FL2-W X FL2-A Y OK 7. OK OK Demodata.001 DNA FACS200 Handbook Pro 15
16 8. Auto DNA non-linear least square analysis 9. Diags Fit Diagnostic %CV, Cell #, RCS, % BAD Poor/ Fair/ Good/ Abnormal %CV G0/G1 CV <8% CV S Cell # RCS %B.A.D 20% 10. OK Error Analysis dialog OK Fit Diagnostics dialog 11. Save Print File Open 2. DNA FL2-A 3. Gate 1 2 Define the Gate X Y OK 4. Mod Help Select Model OK FACS200 Handbook Pro 16
17 > Fresh/Frozen / > Diploid Aneuploid One Aneuploid Two A Tetraploid > No Aggrogate/ aggrogate Auto-Detect > Visible/ Indistinct G2-M G2/M G2/M G0/G1 > Fit Apoptosis/ No Apoptosis DNA Apopsis Sub G1 > No Standards One Standard Two Standards DNA DNA > 5. Range Range Range A1 G0/G1 A1 G0/G1 A2 G2/M A2 G0/G1 G2/M D1 G1/G1 D1 G0/G1 Range D2 G2/M D2 G2/M G0/G1 E1E1 E2 E2 T1 DNA T1 Apop Sub-G1 7. Range Fit non-linear least squares fit 8. Range FACS200 Handbook Pro 17
18 5.1 Status CELLQuest Cytometer Not Ready Ready RUN Standby Standby Run Standby Falcon 2052 Bal Seal Bal seal N0T READY STANDBY RUN READY STANDBY FACS200 Handbook Pro 18
19 Bal seal PRIME RUN STANDBY System Status STATUS READY Instrument Settings CELLQuest Cytometer Status FACSCalibur PRIME O FACSCalibur BD FACS200 Handbook Pro 19
20 1 DNA ACQ R1 M1 File: Gate: G1 Gated Events: 0 Total Events: 0 Marker Events Mean CV All 0 *** *** M1 0 *** *** FACS200 Handbook Pro 20
21 Cell Cycle Analysis Using ModFit LT Number Channels Date acquired: 18-Jan-1995 File: Samplelm.fcs Source: samplelm.fcs Case: PATIENT ID Analysis type: Automatic analysis Prep: Fresh/Frozen DIPLOID: % Dip G0-G1: % at Dip G2-M: 9.49 % at Dip S: % G2/G1: 1.97 Dip %CV: 2.24 Total S-Phase: % Extra Pop: % Debris: 1.18 % Aggregates: 0.00 % Modeled Events: RCS: Diploid B.A.D.: 0.58 % no aggs S-Phase Assessment: Tissue Type: Breast Model type: Diploid Diploid S: High Calculated p-value: p<0.01 S-phase Boundaries: 5.1 and 10.0 FL2-A R FL2-W ModFitLT V2.0(Win32) FACS200 Handbook Pro 21
22 DNA Single Color Two Color, FITC/ PI Threshold FL2-H FL3-H Value FSC PreAmp E00 (Lin) E00 (Lin) SSC Voltage 350 (Lin) 350 (Lin) FL1 Voltage (Log) FL2 Voltage 400 (Lin) - FL3 Voltage (Lin) FL2-A Amp Gain FL3-A Amp Gain FL2-W Amp Gain FL3-W Amp Gain FL1 FL2 0.0% 0.0% FL2 FL1 0.0% 0.0% FL2 FL3 0.0% 0.0% FL3 FL2 0.0% 0.0% BD Falcon 2052 BD Falcon 2052 Falcon 2235 FALCON BD Falcon 2235 FACS Flow 20 BD FACS Clean 5 BD FACS Rinse 5 BD Mesh Filter Hydro-Bios 55µm FACS200 Handbook Pro 22
BD DNA QC Particles RESEARCH APPLICATIONS
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