Workflow Overview. BD FACSDiva Software Quick Reference Guide for BD FACSAria Cell Sorters. Starting Up the System. Checking Cytometer Performance

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1 BD FACSDiva Sofware Quick Reference Guide for BD FACSAria Cell Sorers This guide conains insrucions for using BD FACSDiva sofware version 6. wih BD FACSAria cell sorers. Workflow Overview The following figure shows he seps for daily workflow using BD FACSDiva sofware. Sar Up Sysem Check Performance Se Up Experimen Sor 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. See he BD FACSAria User s Guide for deailed insrucions. Saring Up he Sysem Turn on he cyomeer main power. Sar up he compuer, sar BD FACSDiva sofware, and log in. Check fluid levels in he Cyomeer window. Verify ha he appropriae sor seup is seleced and perform fluidics sarup. seleced sor seup If needed, selec a differen sor seup from he Sor menu. Opimize he breakoff. Checking Cyomeer Performance Turn off he Swee Spo and 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. Verify ha he breakoff is sable and urn on he Swee Spo.

2 Seing Up he Experimen Creae a new experimen and apply he Double Discriminaion analysis emplae. Selec in he Browser and selec heigh and widh for he FSC and SSC parameers in he Inspecor window. Righ-click in he Browser and selec Applicaion Seings > Apply. See page 6 for addiional informaion abou creaing applicaion seings. Selec an applicaion seing. Click Overwrie if necessary. Selec Experimen > Compensaion Seup > Creae Compensaion Conrols. Load he unsained conrol ube. Verify ha he FSC, SSC, and hreshold seings are appropriae. View daa in he normal workshees provided. 6 7 Record daa for he compensaion conrol ubes. View he recorded daa and gae he posiive populaions. Adjus he P gae, righ-click, and selec Apply o All Compensaion Conrols. Adjus he P gaes o fi he posiive populaions.

3 8 Selec Experimen > Compensaion Seup > Calculae Compensaion. Rename he compensaion seup. 9 Record pre-sor daa. Use he Workshee oolbar o creae addiional plos and gaes. Specify crieria under Acquision Seup. 0 Creae soring gaes. Forma SSC-H vs SSC-W and FSC-H vs FSC-W plos o display All Evens and adjus gaes o exclude doubles. Creae gaes for soring paricles of ineres.

4 Soring Insall he collecion ubes and opimize he side seams. Adjus values and sliders as needed. Remove he collecion ubes. Se he drop delay using he Accudrop Drop Delay experimen emplae and Accudrop beads. Alernaively, use he Auo Drop Delay feaure o se he drop delay. Bring in he provided experimen emplae. Adjus he flow rae unil he hreshold rae is ~,000 o,000 evens/second. Sar wih he Precision mode se o Iniial and hen swich o Fine Tune. Sor beads and adjus he Drop Delay unil ~00% are sored o he lef. Open your soring experimen and se up he Sor Layou. Make appropriae selecions and enries.

5 Insall he collecion ubes, sar he sor, and monior he sream during soring. Troubleshoo as needed using he following able as a guide. Observaion Cause Recommended Soluion Soring pauses auomaically. Soring sops auomaically. The sample ube is unloaded. The sream is urned off. The wase drawer closes. The deflecion plaes are urned off. A message appears informing you ha a clog has been deeced. If he Drop or Gap values are ou of range, soring pauses while he ampliude is being adjused. The Drop could no be mached auomaically. Wai for soring o coninue. If his happens ofen, sop he sor, urn off he Swee Spo, and refer o he BD FACSAria User s Guide for insrucions on roubleshooing an unsable breakoff. Turn on he sream and see if Drop reurns o he original value. If Drop reurns o he original value, hen se he Swee Spo and coninue soring. If Drop reurns o a differen value bu he breakoff sill looks good, se he Swee Spo, opimize he drop delay, and coninue soring. If he sream is unsable or leaking, follow he direcions in he BD FACSAria User s Guide o clean a clogged nozzle. When finished, se he Swee Spo, opimize he drop delay, and coninue soring. 6 Sop he sor and save a copy of he sor repor. Analyzing Daa Record pos-sor daa. Check he sor puriy. 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. Specify where o save he PDF and expored saisics files. Shuing Down he Sysem Perform a fluidics shudown or clean he flow cell. Perform exernal cleaning. Turn off he cyomeer main power and shu down he compuer.

6 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. Righ-click he global workshee and apply he Area Scaling analysis emplae. Delee unneeded parameers and selec heigh for he remaining parameers. Forma plos and saisics on he global workshee as needed. Load he appropriae ubes and opimize area scaling values. Adjus he area scaling values unil he area signal maches he heigh signal. Clear he heigh checkboxes for all parameers. Righ-click in he Browser. Selec Applicaion Seings > Creae Workshee. 6 7 Load he unsained conrol ube ono he cyomeer. Adjus he cyomeer seings. Adjus FSC and SSC seings o place populaions on scale and adjus he hreshold. 6

7 8 Acquire 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. 9 Righ-click in he Browser. Selec Applicaion Seings > Save. Rename he seings. 7

8 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. 8