work instruction code version 02 cytometry core

Cytometry Core

Work Instruction Code

ZE5 Version 02

Application Date: 12/05/2020 Standard Operating Procedure Biorad ZE5 of 25

Review Process

Name Function/Unit Date of effective review

C. Guerin Head of unit 12/05/2020

A. Viguier Engineer 24/04/2020

L. Guyonnet Research Engineer - Referent 1 12/05/2020

A. Chipont Engineer - Referent 2 25/03/2020

S. Grondin Engineer 24/04/2020

Thank you to fill in this table with the date after reviewing the document

Synopsis

1 Objective and Scope ................................................................................................................................... 2 2 Authorities and Responsiabilities ............................................................................................................... 2 3 Ressource training ...................................................................................................................................... 2 4 Resource booking ....................................................................................................................................... 2 5 Description ................................................................................................................................................. 3

5.1 Sheath and waste exchange .......................................................................................................... 3 5.2 Start-up .......................................................................................................................................... 5 5.3 Cleaning the fluidics ....................................................................................................................... 5 5.4 Performance Check ........................................................................................................................ 6 5.5 Running Sample ............................................................................................................................. 6

5.5.1 Create a new experiment .......................................................................................................................... 6 5.5.2 Open a previously saved experiment ...................................................................................................... 16

5.6 Data acquisition. .......................................................................................................................... 17 5.7 Shut-down .................................................................................................................................... 20 5.8 Troubleshooting ........................................................................................................................... 21 5.9 Management of sample ............................................................................................................... 21 5.10 Reagent and equipement ............................................................................................................ 22 5.11 Contact details for intervention ................................................................................................... 22 5.12 Technical limitations and constraints .......................................................................................... 22

6 Management of data and metadata .......................................................................................................... 23 7 Definitions/Abbreviations .......................................................................................................................... 24 8 Revision History .......................................................................................................................................... 24 9 Laser configuration ..................................................................................................................................... 24

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1 Objective and Scope

This work instruction describes the procedure for working with the Biorad ZE5 analyzer within the services of the Cytometry Platform of the Institut Curie. The scope is to ensure the proper function of the Biorad ZE5 analyzer and to ensure the safety of users and their samples. This instruction describes the methods of start-up, cleaning the fluidics, exchange sheath fluid and waste, performance check, shut-down, troubleshooting, management of data and additional information.

2 Authorities and Responsiabilities

This work instruction and all related documents have to be followed by all those intending to work with the Biorad ZE5 analyzer of the Cytometry Platform. This concerns the core resource facility users (project leader and research personnel of the IC as well as external academic or industrial users) and the Cytometry Platform personnel. Before operating the Biorad ZE5 analyzer, the user must receive an appropriate training from authorized CPPIC personnel. The instrument is located in the Curie Cytometry Platform in Paris, 26 Rue D’Ulm, Hospital, 6th floor, room 6A11/12 with serial number 820 BR 1123.

3 Ressource training

In order to be trained on the ZE5:

- Go to : http://iris.science-it.ch/ - Create an account if needed - In Scheduler, Timeline, choose flow cytometer Analyser :

- Look for ZE5

- Click on the « Manage » icon - Select « Trainings » tab and click on « Training request » - Fill the pop-up window and click on submit your request.

Once your request has been made, we will send you an invitation for the next training planned on the machine.

4 Resource booking

In order to book a slot to use the instrument:

- Go to http//iris.science-it.ch/ - Your booking rights will be activated once you have done a training on the instrument. If

you are not trained, please submit a training request (cf. chapter 3) - In Scheduler, Timeline, choose flow cytometer Analyser :

http://iris.science-it.ch/

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- You will have access to the schedule of the machine.

- Click on the slot wanted and fill the pop up window and click on “book”.

If you need some help for your experiment, please first warn us and specify on your booking by selecting in booking type: "operator assisted".

5 Description

5.1 Sheath and waste exchange

Before to turn on the instrument or before to run an experiment, check the level of fluidic. To do so, on the left side of the instrument, open the door (figure 1), there are 2 sets of tanks composed of 1 tank of waste (red cap) and 1 tank of sheath (blue cap) each. Each set has an autonomy of 3 hours, be sure that one of the sets is operational (Sheath tank full and waste empty) before to start your experiment so if needed:

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- Empty the waste tank in waste cubitaner "liquides non organiques non halogénés" (red one). When reinstalling waste tanks, make sure you match the dots present on connections.

Wastes are collected and evacuated following the H&S procedure (HSE-10-001-01 Gestion déchets non halogénés Paris).

- Fill the sheath tank with deionized water. When reinstalling sheath tank, probe tip must be far left bottom end of tank (Figure 1, right panel)

Figure 1

If the computer is on and the software running, you can check tanks levels on the Home page (figure 2). The set on run is the one with the glowing green light behind. If you want to switch set of fluidic to have access to the set currently running, click on the “Fluidic swap” icon.

Figure 2

Set 1

Set 2

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5.2 Start-up

The ZE5 hardware is always on, you don’t need to start it up. You only need to start the computer and the software.

1. Turn on the computer and login to Windows with : ID: biorad Password: ZE5265

2. Start Everest software by double-clicking the icon on the desktop. 3. Click on the "startup" icon on home screen (figure 3). 4. Once the cytometer is started, log in the users sessions:

User : users Password: users

Figure 3

5.3 Cleaning the fluidics

If you are the first user to turn on the machine, the following procedure is mandatory:

- On Home page, click on STAT Tube. - Open the sample loader door by clicking on the green button located on the right side of the

loader door on the machine. - Place a 4ml tube of Contrad 10% on the STAT tube holder located on the left side of the plate

loader, close the door. - In Everest software set the flow rate manually at 3.5 µl/secondes (A) and click on play (B).

Let the tube run 3-4min.

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- Stop the acquisition and remove the Contrad 10%. - Place a 4ml tube of Facs Rinse on the STAT holder, close the door, set the flow rate manually

at 3.5 µl/secondes and click on play. Let the tube run 3-4min.

Between users, the following procedure is mandatory: - In tubes, set 1 tube to 200 ul of Contrad 10% and 1 tube to 300 ul of BD FACS Rinse solution. For each Set the flow rate manually at 3.5 µl/secondes. - In 96 wells plate, set 2 wells of 100 ul of Contrad 10% and 3 wells of 150 ul of BD FACS Rinse

Solution. Set the flow rate manually at 3.5 µl/secondes.

5.4 Performance Check

QC bead bottle is permanently on the cytometer. Click on QC button on Home screen. The calibration is automatic. You must have the message: QC successful. If not, refer to the cleaning procedure described on the chapter 5.8 Troubleshooting.

5.5 Running Sample

To analyse your data, you can create a new experiment or you can work with a previously saved experiment.

5.5.1 Create a new experiment

On home page click on new experiment (figure 4).

3.500 A

B

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Figure 4

- Enter a name in the experiment box as : " date name description" (A) . Click (B).

Figure 5

1. FLUOROCHROMES CHOICES - Select the fluorochrome used in your experiment (figure 6)

o Type fluorochrome name to find it in the list (A) and double click on it to select it.

o If you don’t find the fluorochrome in the list, you can manually activate the detector by checking the box in front of the detector of interest (B).

- Type the antibody informations and fluorochrome name, eg : pc7 cd45 (C) if needed. You can also fill in this part a little further on

a)

b)

A

B

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Figure 6

- Click on the right bottom of the screen.

2. PLATE DESIGN - Choose the media (holder) of your experiment (figure 7). You have multiple choices:

o Rack of 24 tubes: 1,5 mL or 2 mL tubes with caps o Rack of 40 tubes: 1,5 mL or 2mL tubes without caps or 5 mL tubes o 96 wells and deep wells plate.

96 wells plates can be round or V-bottom shaped o 384 wells plate.

Figure 7 For the 96 wells plates, you can select predefined media (96 Well Plate) or create a custom type (ask the CCPIC personnel for that).

- Click on the right bottom of the screen.

RackforEppendorf

Rackfor5mltube

Rack of 24 tubes

Rack of 40 tubes

A B C

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At this step, you will configure samples/controls in the tube rack or plate (figure 9). By default, the parameter “Area” is selected for the acquisition (a). If you want to change the media that you are using, you can do it by clicking on the switch media icon

Figure 9

a) Set up of controls for compensation: - To create compensation control tubes positions in the plate setup, toggle the

“compensation control” button to ON (c). - Add an unstained control by toggling the “negative control” button to ON (b)

o Single controls and negative control have now automatically been added for each fluorochrome choosed in the plate map. Compensation controls are in dark green (d).

o Compensation wells are automatically placed. If they are not in the right order, use the drag and drop (e) function to position them at your convenience.

b) Setup your experiment (figure 10)

On the left side of the Plate Setup panel, you can find the experiment name and the panel name. In this menu, you can:

- Set the Run-list Order (a) (horizontally, vertically, etc…) - Set the temperature of the plate holder (b) (from 4°C to 37°C) - Set a Sample line Flush cycle (c), the probe will be entirely (inside and outside) flushed

every x samples: o Highly recommanded if you have “dirty” sample (tumor, skin, dissociated

tissus,…) Check the box to activate the fonction Choose the frequency and the Flush Speed Load a PBS tube (at least 2 mL) on the STAT holder in the machine

- Choose to Shut-Down at the end of your experiment (d) o You must be sure that you are the last user of the day o You must have the cleaning steps included in your experiment

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Figure 10

c) Setup your plate (figure 11) To apply the same settings to all samples, select all of them. Each tube can have its own properties if necessary. Choose the type of well (A):

- Select the wells corresponding to your samples and click on “Sample” o To select a well, click on it. To select multiple, adjacent well, drag over them. To

select multiple nonadjacent wells, press and hold Ctrl and click the wells. o If you want to remove a sample, select the well and click on the trash icon at the

upper right of the Plate Setup Window. - Select the wells corresponding to your washing wells and click on “Wash”.

o In this case no FCS files will be generated. o As mentionned in chapter 5.3 “Cleaning the fluidic”, you need to include washing

spots in your experiment. If you don’t have enough space, you will need to come back after your run to perform the washing steps.

- For all (Setup, Samples and Wash positions), select acquisition limit criteria (B): o On "events" : allow you to record the number of events in total (debris included). o On "volume" : allow you to record a volume in µl (details for Wash in chapter

5.3 “Cleaning the fluidics”) o On " gate" : allow you to record the number you want within the gate that you

will create in the next step. The gate option doesn't work on the compensation control.

- If agitation is required, select wells to be agitated and toggle Agitation button to ON (C) o By default, the agitation lasts 5 secondes (minimum), you can increase the

duration o All the plate is agitated not only the well.

- For all (Setup, Samples and Wash positions), enter a flow rate (D). o There are two possibilities: a volume per seconde or a number of event per

seconde. The flow rate based on volume is more reliable than the one based on events, we recommand to use this option.

- Select all your occupied position and the toggle “Return sample” button to ON (E). The sample pump runs backwards to return any remaining sample to its tube or well. It will

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avoid losing your sample after the Setup step just before acquisition and also to get back your remaining sample after run if needed.

- If you want that the resulting compensation matrix is writen to FCS Files, set a pause on the last compensation control position so that compensation can be calculated before returning to sample recording (F).

- Other option are available if needed: o Wash : cleaning the needle between each sample; 3 options : quick, normal (by

default), full. o High Throughput mode: This mode is very useful for screening. Multiple sample

occupied the sample line at the same time. Be carreful, in this mode you cannot return the sample. Acquisition limit is based on “volume” only.

o Volumetric counting : adds a stabilisation time just after the boost to improve the volumetric counting.

o Reagent : To add reagent to sample. Ensure that at least one position in the plate map is designated as a

Reagent position. Select the sample position to which you wish to add reagent. Click the reagent toggle. Select the position from which to add reagent in the dropdown menu. Specify the reagent volume box. To instruct the system to mix the reagent by asperating and dispensing

the combined sample and reagent several times, select the mix checkbox.

Figure 11

On the right side you can fill in the name of each tube/well:

- Single Menu (A): fill in one by one - Multiple Menu (B): you can add “components” to id your sample or copy/paste (Import)

a list from Excel file.

A

B

C

D

E

F

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- Click at the right bottom of the screen. It will take you to the workspace.

3. WORKSPACE PAGE (figure 13). Each single-color compensation and negative control position already have pre-defined workspace that you cannot modify. For samples you can create your plots and gates at this step, but you can still remove or add some new afterwards. You have only one workspace page per experiment so if the workspace is modified for one sample it will be for all. In the Run-List window, on the left, you can type the antibody and fluorochrome name.

Figure 12

Multiple possibilities are available to display your data. You can create dot plots, histograms and time plots

- Create density plots or histograms (figure 13).

A

B

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o Click on Density Plot (left panel) or Histogram icon on the toolbar (right panel). o Choose the X-axis and Y-axis parameter for dot plot and X-axis for histogram (parameters

are listed under each laser). o For each parameter, you can choose the display (click on the icons, once activated, icons

become blue): Logarithmic scale (log) (fluorescence) if not activated data will be display in Linear

scale (morphology parameters, DNA intercalant for cell cycle, etc…) Hyperlog scale (Hyperlog) Compensated data (comp) Pulse Measurement: Area, Height and Width (area is the one to choose except for

the doublets exclusion and when you are doing cell cycle measurment)

Figure 13

- Create a time plot (figure 14). o Click on Create time plot icon in the toolbar. o Choose the y-axis parameter (Parameter are listed under each laser). o For each parameter, you can choose the display (click on the icons, once activated, icons

become blue): Logarithmic scale (fluorescence) if not activated data will be display in Linear scale

(morphology parameters, DNA intercalant for cell cycle, etc…) Compensated data (comp) Pulse Measurement: Area, Height and Width (area is the one to choose except for

the doublets exclusion) o Select time range for the x-axis.

Sliding : As time progresses, display range remains the same but slides to reflect current time.

Continuous : As time progresses, time range increases to reflect the entire acquisition time.

Fixed : Time range reflects only a fixed maximum, depending on the value that you enter into box.

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Figure 14

If needed, you can also create histograms for all parameters by clicking on the “Add All” icon on the toolbar. Choose the lasers and the display of the parameters.

- Create gates, modify Dot Plot and Histogram using tools (figure 15). After creating a plot or histogram, you can modify it using the tools that appear on the right when you point at the plot or histogram.

o Display a previous gate (a). Allows you to select and display in the current dot plot/histogram a gate/region already created in another dot plot/histogram

o Create a gate using different shapes (b, c, d, e): Rectangle, Quadrant, Polygon, Elipse o Modify Plot Parameter. Allows you to modify x-axis and y-axis such as parameters, scale,

compensation, height, width, area (f). o Add statistics (g). o Add Diagonal Separation (h) Add diagonal line to the plot. This graphical element can help

in setting PMT voltage for compensation when manual. o Track Region Assignement. Allows you to specify target event percentage for a region

within a plot. If the event percentage within the region does not reach the target. Acquisition pauses. Useful as a clog detection tool when utilized with the scatter gate. Applies only to density plot (i).

o Add Anotation within the graph(j).

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Figure 15 If you choose to assign a gate limit of cells to a region, you must do that in the workspace page. There are two ways to do it:

- Create the gate of interest 1. In the plot tool that appears on the side when you pass on the dot plot, select gate limit

assignement icon, check the wanted gate and enter the desired cell number (figure 16.1).

Figure 16.1

2. In the upper tool menu, select the wanted gate in the region drop down menu and enter the desired number of cells. Then, click on sample if you want the rule to be apply on all samples or on selected if you want to rule to be apply on selected sample (Figure 16.2).

Figure 16.2 Once everything is set up in the workspace space, you can click next to access to the acquisition page (cf. Chapter 5.6)

II)

III)

I)

IV)

V)

VI)

(a)

(b) (e)

(d)

(c) (f)

(g)

(h)

(j)

(i)

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5.5.2 Open a previously saved experiment

If your last experiment is recent enough, it can be found in: Recent Experiments on the right of Everest Home Page. You can make new experiment with the same setting, same acquisition page and plate map.

- Check on the menu on the right of Everest Homepage if you find your experiment - Click on (Figure 17):

o Edit if you want to change something in your experiment. It will bring you to the Name Page, all you have to do is to continue like you're doing a new experiment (cf. Chapter 5.5.1). It will keep all your settings, acquisition page and map plate. You can change what you want (add a fluorochrome or delete one etc...)

o Resume : Loads the run list, as it as acquired, into the workspace. Previously acquired positions are indicated in the plate map. This option is useful if acquisition was interrupted in the middle of the previous experiment and also if you want to export Datas.

o Run : Loads a fresh copy of the prevously created run list into the acquisition workspace. This option creates another session in the experiment.Useful if the plate map is exactely the same.

o Import : creates a new experiment (you can't create a new session). Copies fluorophores, parameter names, instrument settings, plots, and the compensation matrix from the run list into the news experiment. It does not copy sample positions.

Figure 17

If your experiment is not in the list of recent experiments (figure 18):

- In Everest Home Page click on Load Experiment (a). - Click on Browser (b), select your experiment's template in D: \user template, select the user session

needed and click OK in the window. o The experiment that you need is the one with run list information as well as telemetry, not

only .fcs files - Click OK (c) .

It will bring you to the Name Page. It will keep all your settings, acquisition page and map plate.

d)a) c)

b)

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Figure 18

5.6 Data acquisition.

When workspace is complete Click on apply (figure 19).

Figure 19

This will take you to acquisition page where you can adjust PMT voltages and acquiring data. There are two modes for running samples: . You choose one of them by clicking on it. SETUP MODE (figure 20). It's the manual mode acquisition. You can adjust the PMT voltage (number or sliders (A)), trigger (B) and threshold (C) in order to set everything right for the acquisition mode (Figure 20.1). You can add up to two trigger (morphological or fluo) by clicking on “2” and choosing the parameter in the drop down menu (D).

ClickApply

h)b)

c)

d)

e) f) g)

j)

a)

i)

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Figure 20.1

When you use setup mode in this way, sampling does not stop unless you instruct the instrument to stop. You can use the record function to record data files from samples. To start the sampling in SETUP MODE (Figure 20.2):

- Choose the sample you want to run by selecting it on the plate plan on the Instrument control (A). - Click on Play (B), the probe will move to the selected position and run sample (click on Pause run

list to pause sampling (C)). - If needed adjust the PMT of the parameter of interest in the PMT Control panel (Figure 20.1) - If needed adjust the trigger parameter (Figure 20.1).

o By default, Forward scatter (488/10) is the only trigger parameter o You can add another one (fluo or morphology)

- If you want to record manually your sample, click on Record (D) (sampling stops when the preset limit is reached.)

While acquiring, you can refresh data displayed on plots in the workspace (E) (Clear data) and establish an update of the data in the dot plot based on the time period (F) (Cycle mode). You can also adjust the Flow rate can either be determined by a target volume or by a target event rate (G). In this mode, you have access to different option:

o Agitate : Click to turn on agitation, click again to turn off (H) . o View run list : Expand the run list to display settings for the current experiment (I). o Edit : Edit experiment. Send you through the experiment Builder to modify the experiment

as needed (I). o Limit : Acquisition limit for record function. To specify an event or volume limit for

recording data in setup mode (I).

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Figure 20.2 ACQUISITION MODE (figure 21). When settings/verifications are done, you can switch to the Acquisition mode (a). You’ll have access only to the “Run” icon and to the flow rate.

- Select the tube/well with witch you want to start the acquisition (b). By default, sampling begins at position A1.

- Click play to record (c) . You will acquire samples in the run list that you set up before.

Figure 21

- If you set a pause on the last compensation control, the run list will stop and let you calculate the compensation matrix. See follow to apply a compensation matrix to your files.

c)

a)

b) b)

c)

a)

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o Click analyse in the Quick Actions area of the toolbar to move the data to the analyse tab. Everest displays the worspace and all plots for all data files.

o Check if negative and positive population are correctly positioned for each compensation control, readjust them if necessary by moving the positive and negative gates.

o Once you are done (Figure 22): Click on compensation's arrow (a). Select prevent Automatic Region Determination (it avoid to go back to the default

gate position) (b). Calculate (c).

Figure 22

o Click Send to Instrument to send the calculated compensation values back to the instrument and to continue running samples with the calculated compensations.

You can acquire your sample with or without compensation as long as your PMT are well setted.

If you acquire with compensations, you can modify them In Everest or in FlowJo.

- To resume the run - Re-start the plate at the first sample after compensation controls by selecting the well and click on

“Run”.

At the end of each experiment, clean the fluidic (see 5.4).

5.7 Shut-down

Shutdown the ZE5 at the end of each day. The shutdown process is entirely automatic and does not require user intervention. You can have planned it on your experiment if you are the last user of the day (Toggle the “Shutdown when complete” button to ON during plate setup) or Click shutdown (in the toolbar or on the Home Page) (figure 23), the system asks you whether you want to schedule an automatic startup, select NO.

I)

II)

III)

a)

b)

c)

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Figure 23

5.8 Troubleshooting

1. QC failed

Re-run the QC, if QC still failed: a) Clean the fluidic (see chapter 5.3) b) Do a clean sample line and a unclogged (figure 24)

If the QC still failed ask the CCPIC personnel.

2. No or low events rates a) Clean the fluidic (see chapter 5.3) b) Do a clean sample line and a unclogged (figure 24)

Figure 24

3. The software is frozen a) If the software freezes during an acquisition click on the icon Resume on the Home Page.

It will bring you to your latest experiment and the latest tube. On acquisition mode, click on play to relaunch the acquisition.

5.9 Management of sample

Sample must be processed and stored according to manufacturer's protocols instruction.

Ontheacquisitionpage

OntheHomePage

CleanandUnclog

CleanandUnclog

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5.10 Reagent and equipement

ZE5 Cell Analyzer Bio-Rad, serial number: 820 BR 1123 ZE serie QC beads Bio-Rad, Reference number: 12004403 ZE5 cell analyzer additive Bio-Rad, Reference number: 12004271 ZE5 cytometer cleaner Bio-Rad, Reference number: 12004272 BD FACS Rinse Solution Becton Dickinson, Reference number: 340346 CONTRAD: DECON VWR, Reference number: 148-0324 5.11 Contact details for intervention

Bio-Rad hotline: 00 800 00 24 67 23 Email: [email protected] Serial number: 820 BR 1123 5.12 Technical limitations and constraints

If you’re encountering an issue while using the machine, submit the issue on Open iris. The action will allow the users to be warn as well as the cytometry staff:

- Go to http://iris.science-it.ch/ - In browser, look for “ZE5” - Click on the resource and then click on the “pen” icon on the upper right

- Fill the pop-up window with the requiered informations and click on “submit”

In order to be worn if users submit an issue, you can activate the issues notifications:

mailto:[email protected]

http://iris.science-it.ch/

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- Click on the ressource and then click on the bell icon in the upper right

- Select “notify me issues” and close.

6 Management of data and metadata

DATA EXPORT - Data export is under responsibility of the user At the end of the acquisition, Click on Export Data icon in the acquisition toolbar.

Figure 25

- Click on Export on the botton right of the screen o In the Save As dialog box, browse to a location : D:\Everest\USERS FCS FILES. o Create a new file named “YYMMDD_Yourname_description”

- Click on OK - Select the format:

o FCS files : export all FCS files for the current experiment (a). o FCS files to Zip : export FCS files and compress to ZIP (b). o Run list and FCS Files : export run lit and export all FCS files for the current

experiment (c). o Full Experiment to Zip : export full experiment, including run list and FCS files

compress to Zip (d). - Select the paramaters exported (e):

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o SSC and FSC in Area, Height and Width o Fluo in Area only unless you are using the other parameters (i.e. cell cycle) it avoid

heavy files - Once this is done, on desktop double click on "connect to synapse". - Connect to CytometrieFlux server using your ID and pass word - On the desktop there is a shortcut called: USERS FCS FILES from which you will transfer your

data in your folder on CytometrieFlux.

BACKUP YOUR DATA TO SYNAPSE IMMEDIATELY AFTER ACQUISITION Every Monday we will delete run list files and FCS files acquired less than one week older.

DSI is handling back-ups of Institut Curie servers. SAVE A TEMPLATE If you want to keep the settings and page of acquisition of an experience you must save it in the user template file. During the acquisition, your experiment is automatically created and saved here: D: \Everest\Users (there is a shortcut on the desktop). This version of your experiment contains information required to load a previous experiment. If you want to do a new session later from this experiment, copy paste it from D: \Everest\Users (shortcut named user on the desktop) to D: \user template. in folder of your name. Use the shortcut on the desktop to find your experiment.

7 Definitions/Abbreviations

QC Quality Control DI Deionized FCS Flow Cytometry Standard Sec Second ml Milliliter µl Microliters CV Coefficient de Variation

8 Revision History

Version Effective date Summary of change

01 10/04/2019 New document

02 21/03/2020 New version

03 07/04/2020 New Version

9 Laser configuration

ZE5 Cell Analyzer 5 lasers, 27 colors (5/7/4/7/4)

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Fluorochromes list is not exhaustive

work instruction code version 02 cytometry core

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