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.

Laser	Excitation	Emission	Common Dye	Fluorescent protein
Violet	405 nm	440 nm	Pacific Blue	ECFP
Violet	405 nm	512 nm	Pacific Green
Violet	405 nm	603 nm	Pacific Orange
Violet	405 nm	710 nm	Qdot 705
Blue	488 nm	530 nm	FITC	EGFP, Emerald GFP
Blue	488 nm	574 nm	Propidium iodide	EYFP
Blue	488 nm	695 nm	PerCP-Cy5.5
Yellow	561 nm	583 nm	PE	RFP
Yellow	561 nm	620 nm	PE-Texas Red	mCherry, dTomato, DsRed, mStrawberry
Yellow	561 nm	695 nm	PE-Cy5.5
Yellow	561 nm	780 nm	PE-Cy7
Red	638 nm	660 nm	APC
Red	638 nm	720 nm	Alexa Fluor 700
Red	638 nm	780 nm	APC-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.