Transcriptic

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

Transcriptic 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.

Flow Cytometry


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