Workflow Overview. BD FACSDiva Software Quick Reference Guide for the BD LSR II. Starting Up the System. Checking Cytometer Performance

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1 BD FACSDiva Sofware Quick Reference Guide for he BD LSR II This guide conains insrucions for using BD FACSDiva sofware version 6.0 and laer wih BD LSR II flow cyomeers. Workflow Overview The following figure shows he seps for daily workflow using BD FACSDiva sofware. Sar Up Sysem Check Performance Se Up Experimen Record Daa Analyze Daa Shu Down Sysem Before saring your daily workflow, ensure ha your lab s sofware adminisraor has performed all he necessary asks o se up he sofware for your use. This guide shows a workflow ha uses applicaion seings. Saring Up he Sysem Sar up he cyomeer and compuer. Sar BD FACSDiva sofware and log in. Prepare he fluidics anks and remove bubbles from he fluidics sysem. Verify ha he opical filers are appropriae for your experimen. Checking Cyomeer Performance Selec Cyomeer > CST. Verify he Cyomeer Configuraion and bead lo ID. If needed, selec a differen configuraion or bead lo ID. Run he BD Cyomeer Seup and Tracking beads. View he Cyomeer Performance Repor. Close he Cyomeer Seup and Tracking window

2 Seing Up he Experimen Creae Browser elemens. Use he Browser oolbar o add elemens. folder experimen Righ-click in he Browser. Selec Applicaion Seings > Apply. See page 5 for addiional informaion abou creaing applicaion seings. Selec an applicaion seing. Click Overwrie if necessary. Selec Experimen > Compensaion Seup > Creae Compensaion Conrols. Creae label-specific conrols as needed. Insall he unsained conrol ono he cyomeer. Click. Verify ha he FSC, SSC, and hreshold seings are appropriae. View daa in he normal workshees provided

3 5 Record daa for he compensaion conrol ubes. Adjus he P gaes o fi he posiive populaions. Adjus he P gae, righ-click and selec Apply o All Compensaion Conrols. 6 Selec Experimen > Compensaion Seup > Calculae Compensaion. Rename he compensaion seup. Recording Specimen Daa Creae Browser elemens. Creae he plos, gaes, and saisics needed for recording. Use he Workshee oolbar o creae plos and gaes. Righ-click o show he saisics view and populaion hierarchy. Make enries in he Experimen Layou. Specify reagen labels, keywords, and acquisiion crieria as needed. Record daa

4 Analyzing Daa Creae plos, gaes, and saisics needed for analysis. Creae cusom ex and graphics. Creae new global workshees. Cusomize plos using he Plo Inspecor. Perform qualiy conrol of he analysis. Verify ha gaes are se appropriaely for all samples. Use he populaion hierarchy o verify paren/child relaionships. Do one of he following o prin or expor he resuls. Selec File > Prin o prin he acive workshee. Selec File > Expor o expor seleced elemens. Righ-click a specimen or experimen and selec Bach Analysis (using a global workshee). Selec o prin, save as a PDF, or expor he saisics as needed. Shuing Down he Sysem Clean he fluidics. Selec File > Qui. Turn off he cyomeer and compuer

5 Creaing Applicaion Seings Before creaing applicaion seings, perform he cyomeer sarup procedure according o your cyomeer user s guide and run a performance check. Creae a new experimen. Add a specimen. Delee parameers no needed. Se he curren ube poiner. Righ-click in he Browser. Selec Applicaion Seings > Creae Workshee. Insall he unsained conrol ono he cyomeer. Click. Adjus he cyomeer seings. Adjus FCS and SSC volages o place populaions on scale, and adjus he hreshold. 5 Acquire he single-sained conrols. If needed, decrease he PMT volages o place he posiive populaions on scale. Use he gray boxes as a guide when decreasing he PMT volages. 6 Righ-click in he Browser. Selec Applicaion Seings > Save. Rename he seings

6 Cyomeer Seings Overview Cyomeer Seings Icons in he Browser Defaul or user-defined seings have been applied. CS&T defined seings are applied. User-defined applicaion seings have been applied. A compensaion seup has been linked o he applicaion seings. A compensaion seup has been linked o he cyomeer seings. Cyomeer Seups in he Caalog Seing ype Descripion When o save How o save How o apply Wha is saved Parameers, PMTV, and hreshold Area scaling and laser delay** Compensaion Applicaion Seings User-opimized seings for sample ype and reagen combinaion. CS&T updaes hese seings o accoun for daily variaions. Creae once for each applicaion you are running. Verify he seings afer defining he baseline and when you change reagen los. Righ-click he Cyomeer Seings in he Browser and selec Applicaion Seings > Save. Righ-click he Cyomeer Seings in he Browser and selec Applicaion Seings > Apply. Selec an applicaion seing from he caalog. * Compensaion Seup Seings ha include auomaically calculaed compensaion values creaed by BD FACSDiva sofware using single-sained conrols. Creae daily for each experimen. Selec Experimen > Compensaion Seup > Calculae Compensaion. In he Single Sained dialog, selec Link & Save. Righ-click he Cyomeer Seings in he Browser and selec Link Seup. Selec a compensaion seup from he caalog. No updaed by CS&T. Cyomeer Seings User-opimized seings, no updaed by CS&T or linked o a compensaion seup. As needed. Righ-click he Cyomeer Seings in he Browser and selec Save o Caalog. Righ-click he Cyomeer Seings in he Browser and selec Apply from Caalog. Selec a cyomeer seings from he caalog. * The area scaling is adjused for he CS&T beads only. You may need o readjus his seing for your cell ype. ** The laes opimized laser delay seing is always used on he cyomeer. When he saved cyomeer seings are re-applied, he laser delay does no change o he value saved wih he seings. The laser delay seing is saved for reference only