Flow Cytometry

Instruction and Parameters

The flow_analyze instruction takes parameters to modify and add channels from a list matching the instruments capabilities. For any channel you will have to specify which parameters to collect (area,height,width) and you can suggest a voltage_range in which we check for the best resolution and data distribution of your samples. The actual voltage used will be returned to you in the results.

In addition to general set-up you need to specify at least one negative control and one sample. The negative control is used to set-up the experiment and optimize the voltage for that given channel/color. You may have more than one negative control. Positive controls in this instruction refer to technical positive controls, which may overlap with your biological controls. They are used to determine the maximum expected signal on their given channel, as well as any color bleed that can be later corrected by compensation.

Only after all negative and positive controls have been run will the samples be analyzed. A well can be used for both control and sample, assuming enough liquid is present.

Any data collection step will be run until the specified volume is run or the number of captured_events is reached, whichever occurs first. Please note that volume is a required parameter and captured_events is optional.

{
  "op": "flow_analyze",
  "dataref": "flow_data",
  "channels": {
    "FSC": {
      "voltage_range": {
        "low": "230:volt",
        "high": "280:volt"
        },
      "area": true,             //default: true
      "height": true,           //default: true
      "weight": false           //default: false
      },
    "SSC": {
      "voltage_range": { … },
      "area": true,             //default: true
      "height": false,          //default: false
      "weight": false           //default: false
      },
    "colors":[{
      "name": "FitC",
      "emission_wavelength": "495:nanometer",
      "excitation_wavelength": "519:nanometer",
      "voltage_range": { … },
      "area": true,             //default: true
      "height": false,          //default: false
      "weight": false           //default: false
    }, … ]
  },
  "negative_controls": [{
    "well": well,
    "volume": volume,
    "captured_events": integer,     // optional, default infinity
    "channel": [channel_name]
  }],                               // at least 1 negative control req'd
  "positive_controls": [{
    "well": well,
    "volume": volume,
    "captured_events": integer,     // optional, default infinity
    "channel": [channel_name],
    "minimize_bleed": [{            // optional
      "from": color,
      "to": [color]
    }, … ]
  }],
  "samples": [{
    "well": well,
    "volume": volume,
    "captured_events": integer,     // optional, default infinity
  }]
}
{
  "parameters": {
    "FSC_voltage": volt,
    "SSC_voltage": volt,
    "colors":{[
      "name": string,
      "actual_voltage": volt
    ], … },
  }
  "data": {
    "plate1/A3": {
      "fsc_3.1": "url"
    }
  }
}

Supported excitation and emission wavelengths

Use any of the following combinations of excitation and emission wavelength. The name can be chosen freely and we recommend to use the name of the dye or fluorescent protein that you are detecting.
The name chosen here will be used to determine any specific negative and positive controls.

LaserExcitationEmissionCommon DyeFluorescent protein
Violet405 nm440 nmPacific BlueECFP
Violet405 nm512 nmPacific Green
Violet405 nm603 nmPacific Orange
Violet405 nm710 nmQdot 705
Blue488 nm530 nmFITCEGFP, Emerald GFP
Blue488 nm574 nmPropidium iodideEYFP
Blue488 nm695 nmPerCP-Cy5.5
Yellow561 nm583 nmPERFP
Yellow561 nm620 nmPE-Texas RedmCherry, dTomato, DsRed, mStrawberry
Yellow561 nm695 nmPE-Cy5.5
Yellow561 nm780 nmPE-Cy7
Red638 nm660 nmAPC
Red638 nm720 nmAlexa Fluor 700
Red638 nm780 nmAPC-Alexa Fluor 750

Device

Strateos uses the Attune NxT Acoustic Focusing Cytometer equipped with a violet, blue, red and yellow laser. In addition to sheath fluid the Attune uses sound waves to focus your cells, allowing for fast acquisition speeds while maintaining high resolution.
The instrument is routinely run at 100uL/min, with flow rates being adjusted to reflect sample quality and complexity.

Before analysis in the system, every well is mixed 2 times to suspend the cells properly. The sample is not recoverable, meaning if captured_events is set and this limit is reached before the entire volume of cells has been analyzed, the remaining volume is discarded and not returned to the source plate.