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4 

 

Suggestion for “Materials and Methods”

 

Flow cytometry data were acquired on a Bio-Rad S3e Cell Sorter and analyzed using 

the ProSort software, version 1.6. 

Flow cytometry data were acquired on a Bio-Rad S3e Cell Sorter operated by the 

ProSort software, version 1.6, and analyzed using the Cytospec software from Purdue 

University Cytometry Laboratories.  

Bacillus subtilis 

cells were sorted as alive GFP and dead GFP cells using a Bio-Rad S3e 

Cell Sorter operated by the ProSort software, version 1.6. 

 

Suggestion for “Acknowledgements”

 

This work was partially supported by PPBI - Portuguese Platform of BioImaging (PPBI-

POCI-01-0145-FEDER-022122)  co-funded  by  national  funds  from  OE  -  "Orçamento  de 

Estado"  and  by  european  funds  from  FEDER  -  "Fundo  Europeu  de  Desenvolvimento 

Regional

”.

 

 

People and Contacts 

Adriano O. Henriques, Microbial Development Group (

[email protected]

)  

Mónica Serrano, Microbial Development Group (

[email protected]

) 

Carolina Cassona, Microbial Development Group (

[email protected]

) 

Fernando Cruz, Microbial Development Group (

[email protected]

)  

Phone +351 214 469 524 

Extension 1524 

 

 

 

Содержание S3e

Страница 1: ...Flow Cytometry FACS Bio Rad S3e Cell Sorter...

Страница 2: ...7 WARNINGS and Advice 8 Available Techniques 9 Set Up 9 Sample Preparation 9 Initial Set Up Start up and Fluidics Procedure 20 minutes 10 Initial Set Up Quality Control 10 minutes 12 Final Set Up Low...

Страница 3: ...lex presort setup with a unique technology ProDrop technology In detail this cell sorter brings forward several automated features such as Startup shutdown Stream to laser alignment Hands free alignme...

Страница 4: ...l Sorter operated by the ProSort software version 1 6 Suggestion for Acknowledgements This work was partially supported by PPBI Portuguese Platform of BioImaging PPBI POCI 01 0145 FEDER 022122 co fund...

Страница 5: ...cy 37 43 kHz Sorting type True jet in air for high performance sorting Sorting rate No hardware limitations for sort rate limited only by droplet frequency and application Sorting purity 99 pure Nozzl...

Страница 6: ...6 565 554 578 584 FL3 BP 615 25 PI PE Texas Red 536 496 565 617 619 FL4 655 LP PE Cy5 PE Cy5 5 PE Cy7 496 565 490 565 496 565 670 693 785 561 FL2 BP 586 25 PE tdTomato RFP DsRed 496 565 554 554 554 57...

Страница 7: ...Computer Type Desktop Brand Dell Inc Model OptiPlex 3020 Features Intel I5 4590 up to 3 7 GHz quad core 4GB RAM 500 GB HDD OS Windows 7 Professional Available Software ProSort Monitor Brand Asus Model...

Страница 8: ...ube and nozzle tip Do not open the nozzle door Keep the green containment door closed as often as possible during the sorting procedure Never try to access the laser beam paths Any adjustment to the t...

Страница 9: ...70 ethanol and MiliQ water as cleaning solutions Please let us know if some solution or fluidic is running out Make sure to save all the data to your own storage device or cloud as we routinely wipe o...

Страница 10: ...d quality control procedures NEVERTHELESS DO NOT FORGET TO CHECK THE FLUIDICS IF NEEDED PERFORM THE HOT SWAP PROCEDURE See Swapping Fluidics During Sorting Hot Swap 1 Open the bulk fluidics door 2 Det...

Страница 11: ...r a click 9 Switch on the computer 10 Double click in the ProSort icon in the desktop 11 Log in into the group account Contact the responsible people for more log in options 12 In the Setup and Mainte...

Страница 12: ...T SWAP PROCEDURE See Swapping Fluidics During Sorting Hot Swap 1 Vortex a bottle of ProLine Calibration Beads and place 8 10 drops into a 5 ml sorter tube Do not dilute these particles 2 Place the tub...

Страница 13: ...QC procedure estimates the optimal drop delay and performs the side and forward scatter detectors calibration 6 A QC report window will appear showing the QC test results If all parameters have passe...

Страница 14: ...in the Clean System button 4 A Clean Warning dialog will open with a prompt warning for the fact that the tube solution will be rapidly consumed Click in the Yes option 5 In the Clean System Modes win...

Страница 15: ...70 ethanol 2 Load the tube into the sample station and move it to the run position 3 In the Set Up and Maintenance tab click in the Clean System button 4 A Clean Warning dialog will open with a prompt...

Страница 16: ...Click in the Shutdown button in the Setup and Maintenance tab 10 Select the Yes option to confirm that the system should be shut down at this time 11 Select the Yes option to continue without saving p...

Страница 17: ...and Shutdown window load a tube of water MiliQ and move the loading stage into the run position 15 After the cleaning procedure move the loading stage into the wash position 16 At this time the syste...

Страница 18: ...t plot by clicking in the Histogram and Density buttons of the Home tab 3 Load the sample into the sample input station and move the loading stage into the run position 4 Detectors can be renamed for...

Страница 19: ...threshold value 7 In the Instrument Control Panel window click in the Start button to begin sample acquisition 8 Adjust photomultiplier tube PTM voltages to place populations 9 Adjust the target even...

Страница 20: ...fresh button can be used to refresh the data as adjustments are being made 12 Re collect the sample and save the FCS format file if Auto Save has not been boxed 13 Unlock the loading stage and remove...

Страница 21: ...o set compensation for multi coloured samples see Auto Compensation Wizard Sorting 1 Go to File New Protocol or Open Protocol see Protocols and Workspace 2 Create at least one histogram and dot plot b...

Страница 22: ...e dot plot The signal intensity and time should be displayed on the y and x axes respectively Then select as many dots as possible linearly disposed as if in a direct proportion The dots contained in...

Страница 23: ...ight click in the desired region to be sorted and select the sort direction 7 Repeat with a second region if two way sorting is desired 8 Click in the Sort Logic button and select a collection vessel...

Страница 24: ...negative match events advised for low concentrations The Purity sort mode stands for sorting only positive match events although there may be double or more events sorted at once The Single sort mode...

Страница 25: ...ction device at the previously selected positions 12 Make sure the 5 ml tubes if it is the case have at least 0 5 ml of buffer to cushion the sorted cells 13 Close the sort chamber door and click in t...

Страница 26: ...26 14 The Sort Statistics window will appear and sorting will be initiated This window can be reopened by clicking the Sort Statistics button in the toolbar...

Страница 27: ...ore clicking the Swap Fluidics button as the swap time is limited to only 3 minutes before the system starting to shut down due to low fluid levels Read carefully all procedure before starting the Hot...

Страница 28: ...tton 5 Lift the quick disconnect until it engages the magnetic holder This will hold the quick disconnect away from the exchange area 6 Slightly lift and pull out the container 7 Uncap the cap assembl...

Страница 29: ...n single controls for each parameter and your multi stained sample 2 Save the acquisitions as FCS files 3 Go to Home Compensation Wizard 4 Select the parameters for compensation 5 Click the file icon...

Страница 30: ...er negative and positive regions as necessary Note that the positive and negative population gates must not overlap 10 Click the blue forward arrow to go to the next control 11 Repeat steps 9 and 10 f...

Страница 31: ...that have values lesser than zero to be correctly displayed This data display format is used only for compensated data It converts a small portion of the scale around zero to linear and adds a small a...

Страница 32: ...rotocol can also be opened from a saved FCS file see below how to perform this in another way Saving Protocols Each protocol is saved with the following information plots regions gates trigger thresho...

Страница 33: ...Remember that you can save FCS files after running acquisition or sorting procedures In addition the FCS files will not only save the procedures statistics as will also store the protocol in use Furth...

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