BD^® Research Cloud (BD RC) is a collaborative environment to enable flow cytometry experiments. BD RC is designed to integrate and facilitate flow cytometry experiments from panel design to analysis. The cloud-based software allows labs to create customized workflows for tracking collaborative experiments, store metadata in the context of the experiment and design panels.

You can sign up for an account at bdresearchcloud.com. Creating an account is free and does not require any payment information.

Yes, BD RC accounts are supported by FlowJo Portal. We recommend that you sign up for a BD RC account using an existing FlowJo Portal ID if you have one. Changing the password for your FlowJo Portal account will change the password for your BD RC account and vice versa.

If you create your BD RC account prior to creating a FlowJo Portal account, you may need to sign in using the account in order to finish the process of creating the FlowJo Portal  account for some FlowJo™ Software licensing workflows.

When you create a BD RC account, you will be creating a FlowJo Portal account. BD RC and the FlowJo™ Software interface will still work as expected even if you’re not using FlowJo Portal to authenticate FlowJo™ Software.

View the BD RC End User License Agreement and Privacy Statements for details on how BD uses your data. In general, there are three defined types of data and three categories of data usage.

Data Types:

Personal Data—information about a user, including identifying information

• Experimental data—results of tests, analyses or experiments
• Product Data—information related to a user’s reagents, instruments, antigens, antibodies, panels or purchases
  

Usage categories:

• BD RC use—BD uses your data to provide features and functionality within BD RC, such as storage, collaboration and management of your data. This is the only use for experimental data, and this use also applies to product and personal data.
  
• Product Development use—BD uses your product data to further improve BD RC as well as improve or create other product offerings from BD, including reagents, instruments or software.
• Marketing use—BD uses your product and personal data to create marketing material and market to you and/or others about BD product offerings.

View the BD RC End User License Agreement and Privacy Statements for details on how BD protects your data. BD RC is built on Amazon Web Services (aws.amazon.com) and the security they offer.  Cytometer Configuration files are the only files uploaded to BD RC, which are used for Product Development or Marketing uses. All other files uploaded to BD RC are only used for the purposes of supplying BD RC features and functionality.

Your experimental data will be removed after some period, depending on the type of license that your organization had. At no point will BD have access to your experimental data.

Once you are at your data allotment, you will need to upgrade your license or purchase more storage before uploading any additional files. BD RC will not delete any files without your express action, even when you meet your data limit.

BD RC users join an organization, the first level of privacy and security for your data. Then users can form into groups to further control who can view stored files and collaborate on projects. Projects are made up of workflows and the files that are associated with those workflows, while studies are replicates of workflows over time.

Organizations should be shared primarily by people collaborating on flow cytometry experiments. However, groups can also be used to provide data security and privacy from other users in an organization to allow larger institutions to bundle users together for billing purposes.

Reagent Selector is a predefined workflow to guide you through the panel design process. It’s a five- step workflow that helps you select your instrument, define your biology, choose the antibody and fluorochrome pairings, and choose reagents and order them for your experiment. The workflow enables panel design for the markers and subpopulations you define using available reagents that are compatible with your instrument.

Reagent Selector requires two pieces of information:

1. Cytometer configuration—a description of the optical configuration of your cytometer, including laser and filter/detector info. Cytometer configurations may be input manually using the Cytometers resource or you can import the configuration of a BD cytometer using the cytometer configuration file accessible through CS&T. A cytometer only needs to be imported or defined once and will subsequently be accessible to use for panel design by any other BD RC users with access to the cytometer. Where in BD RC: Reagent Selector Step 1
   
   
2. Biological hypothesis—at a minimum, you only need to select the set  of markers you want to include in your panel. To facilitate your panel design process, it is strongly recommended that you add antigen density estimates where possible. Furthermore, if you know the antigen density co- expression patterns for multiple populations of interest in your experiment, you can define multiple subpopulations, each with individually adjustable antigen densities for each marker. Where in BD RC: Reagent Selector Step 2

Additionally, you may provide optional inputs to help meet your panel design needs:

1. Custom reagents—reagents not available in the BD catalog that you would like included as options in the panel design process. These may include reagents that you already have in your lab’s inventory or other non-catalog reagents. Note that Reagent Selector can only be used for reagents based on BD fluorochromes or non-proprietary fluorochromes (see detailed list). Where in BD RC: Reagents resource
   
   
2. Clone preferences—specific clones to use for selected antibodies. Reagent Selector will only list reagents available for the specified clone. Where in BD RC: Reagent Selector Step 2
   
   
3. Viability Dyes—indicate that your panel requires a reagent that distinguishes live and dead cells. Then, available Viability Dyes from the BD catalog and any custom reagents will be available on Reagent Selector Step 3. Where in BD RC: Reagent Selector Step 2

Your cytometer configuration is used to determine which fluorochromes are measurable on your  instrument; the fluorochrome must be adequately excited by at least one laser and its emission must be adequately captured by at least one detector band in order to be included for consideration. The panel size may not exceed the total number of detectors on the cytometer (note that if multiple markers are designated as Exclusion, they will be assigned to the same fluorochrome and therefore only count as one marker against the panel size limit). Your cytometer configuration will also be used to assign fluorochromes to primary detectors and to generate a BD FACSDiva™ Software experiment template, if applicable.

BD^® Research Cloud uses the following pieces of information, so make sure they are as accurate as possible:

• Laser power
• Laser wavelength
• Detector band (determined from BP and LP/SP filters if present)

You may manually define the cytometer by inputting its laser and detector info. Note that for cytometers lacking traditional BP+LP filter arrangements, you may instead input the effective filter band for each detector as a single BP filter.

Public Cytometers are cytometers that are visible to any BD RC user, regardless of their organization. BD RC users are able to link to Public Cytometers outside of their organization for use in designing panels or creating BD FACSDiva™ Software experiments. Public Cytometers are not editable by users in the linked organization, only by the organization in which they are created. If a Public Cytometer is edited and made to be no longer publicly available, then it will be removed from the linked organizations.

Reagent Selector will provide reagents that are available in the BD catalog and custom reagents that have been defined by the user.

You may define custom reagents with the Reagents resource. Note that panel design can only be used with reagents based on BD fluorochromes or non-proprietary fluorochromes (see detailed list below).

Fluorochromes key: BB, BD Horizon Brilliant™ Blue; BUV, BD Horizon Brilliant™ UV; BV, BD Horizon Brilliant™ Violet; BYG, BD Horizon Brilliant™ Yellow-Green; FVS, BD Horizon™ Fixable Viability Stain; R, BD Horizon™ Red; RB, BD Horizon RealBlue™; RY, BD Horizon RealYellow™; V, BD Horizon™ Violet.

*Spectral data is not available for these fluorochromes. These can still be used during panel design, but will not be shown on spectral signature plots or the similarity matrix, and will not be computed as part of the complexity score for a panel.

Fluorescent proteins can be added to a panel by making a Custom Reagent and then adding that custom reagent to the panel. For example, if you have an eGFP reporter for transcription factor FOXP3, create a custom reagent with a custom name (i.e., FOXP3-eGFP) and fill out as much information about that marker as possible—most importantly the fluorochrome (eGFP). When you navigate back to Step 2 of Reagent Selector, you can now add that reagent to your panel by searching for the antigen associated with custom reagent.

BD FACSymphony™ A5 SE Flow Cytometer set up:
Unstained PBMC were used to determine PMT voltage settings. The voltage for each detector was set so that the resulting rSD of the unstained cells was approximately three times the robust standard deviation of electronic noise (rSDEN). The results of the Cytometer Baseline report were used to obtain the electronic noise (rSDEN) for each channel. Once the voltage for each PMT had been established, an application-specific setting was created.

CD4 staining protocol:
Normal human PBMC were stained with anti-CD4 reagents at the manufacturer-recommended concentration; manufacturer-recommended buffers were used during the staining procedure. Cells were stained for 30 minutes at RT, protected from light. Samples were washed three times with wash buffer and immediately analyzed on the BD FACSymphony^™ A5 SE Flow Cytometer.

Brightness scoring:
The brightness score assigned to each fluorochrome was based on stain index (SI) values and calculated using the formula (MFI_positive – MFI_negative)/(2*rSDnegative). MFI values were derived from spectral unmixing across 48 detectors and are specific to the BD FACSymphony^™ A5 SE System configuration, which includes laser wavelengths, laser wattages and instrument settings (PMT voltage). The performance features of this instrument may impact the resolution of certain fluorochromes, and variation in stain index between other spectral or non-spectral flow cytometers is expected.

Antigen density refers to the estimated abundance of marker molecules on the cell in question. Antigen density in BD RC is binned into one of the four expression levels or into Unknown:

• None: Unexpressed—the marker is not present (or is expressed at unmeasurably low levels) on this population. Fluorochromes assigned to this marker will therefore cause no spillover spreading into other markers on this population, but resolution of this marker (spread of the negative) will still be impacted
  
• Low: Expressed at a low level. Fluorochromes assigned to Low-expression markers will cause minimal spillover spreading into other markers, but expression of Low-expression markers will also be maximally susceptible to resolution-damaging spillover spreading from other fluorochromes.
• Mid: Expressed at a moderate level (10x higher than Low). Fluorochromes assigned to Mid- expression markers will both cause and receive spillover spreading at moderate levels.
• High: Expressed at a high level (100x higher than Low). Fluorochromes assigned to High- expression markers will be the most likely to cause severe spillover spreading into other co-expressed markers. However, these markers will also be least affected by spillover spreading into their fluorochromes.
• Unknown: Unknown expression level. You could assume that this marker contributes a moderate amount of spillover spreading (equivalent to Mid) but has minimal susceptibility to spillover spreading into its fluorophore (equivalent to High). Try not to assign markers designated Unknown to fluorochromes that cause overly severe spillover spreading but you can allow a higher degree of spillover spreading into this marker’s fluorochrome.
  

Note that antigen density in BD RC is considered an absolute metric across all populations and markers within a given experiment, i.e., a marker designated as High is assumed to be present in the same abundance on a cell as another marker designated as High. Note that this differs from some conventions wherein antigen density is described only relative to the marker itself (such as CD4-High vs CD4-Low cells and CD25-High vs CD25-Low cells).

Reagent Selector combines all markers designated as Exclusion and allows you to assign them to a single fluorochrome.

Reagent Selector will allow you to select an available viability dye on Step 3 if you indicate a need on Step 2.

Projects can only be created in the Projects & Studies section of BD^® Research Cloud and only by those with the permission to do so. If you want to create your own Project, you will have the permission to do so in your Individual Org or if you create your own Group.

Groups of users are assigned Projects to work on. Projects are like a container or folder to organize Workflows and Studies. Typically, only users within the groups assigned to the projects can view the details of the projects, workflows or studies. However, users with the permission View workflows outside group will be able to see all workflows in the BD RC organization.

Anyone in your Group has viewing access to the Projects assigned to the Group. Users outside the group do not have access unless they have the permission View workflows outside group.

A Workflow is a defined set of procedures and steps designed to capture a single experiment from start to finish. If the experiment is often repeated and metadata is collected during the process, a Workflow can be made into a Study to collect longitudinal data over time. As an example, a workflow could be Setting-Up Cytometer for Daily Use that describes that specific process, but that Workflow could be converted into a Study if it is important to collect Power-On Time or Sheath Tank Level data over time.