CELL TYPES, SIZES & MORE...

Q1. What type of cells can Inish analyse?

A1. T-cells, PBMCs, stem cells, cell lines (Jurkat, CHO etc.), yeast cells and sperm cells.

Q2. What size cells can Inish analyse?

A2. Inish analyses cells in the size range of 2µm - 25µm.

Q3. What is the concentration range of cells that Inish can analyse?

A3. 50,000 - 2,000,000 cells/mL where 40µL of sample is used for each assay.

Q4. Are the different Sample Injector Modules (SIM) based on the cell sizes?

A4. Yes. 

SIM30 is suitable for analysing cells of 2µm - 12µm in size and

SIM60 is suitable for analysing cells of 6µm - 25µm. 

SIMs are interchangeable - so you don't have to buy a whole new Inish Analyser for each cell size range; just swap out the SIM (as shown in "Product Questions?").

Q5. How many cells are needed for one assay?

A5. Minimum number of cells per assay is 2,000

Maximum number of cells counted per assay is 10,000.

This is based on the recommended concentration of 50,000 - 2,000,000 cells/mL where 40µL of sample is used for each assay.

Q6. How do you kill cells for control experiments?

A6. This depends on the cell type and which assay you are conducting.  Contact Cellix for more information.

Q7. Can I use cells that clump a lot?

A7. No - cells that clump will block the instrument.

Q8. After the assay, do you have a pure population of live cells?

A8. No - the Inish analyses the cells but it does not sort them or enrich them. The sample is collected in the waste tube and can be recovered.

Cell types and sizes
 

INISH TECHNOLOGY

Q1. What is the technology used in the Inish Analyser? / What is the principle of detection used?

A1. Impedance Spectroscopy. This is a method of measuring the resistance and capacitance properties of a cell or particle by applying an electric field across two electrodes embedded in a microfluidic chip.  As each cell flows past the electrodes, the electric field is disturbed.  The advantage of this technology is that cells do not have to be labelled or stained to detect changes - it is label-free!

Sample is drawn into the Inish Analyser from the sample tube:

Sample is pumped through the microfluidic impedance chip.  This chip contains embedded electrodes and the cells flow past these electrodes.

As the cells flow past the electrodes, they disturb the electric field.  

This results in a true "single cell analysis" technique where results are reported in standard FCS file format:  every dot on the scatterplot represents a single cell.  

Q2. How often do I have to change the cartridge / microfluidic chip in the SIM?  

A2. This usually needs to be changed once per year. All Inish Analysers are shipped with a SIM box for annual return of the SIM for servicing.  The first service is free and includes a replacement cartridge / microfluidic chip; and recalibration and testing of the SIM.

Q3. Does Inish have a flow rate indicator?

A3. No but... 

We measure the flow rate by measuring the transit time - this is the time it takes for a cell to pass from one electrode to the next electrode in the microfluidic chip.  From this, we calculate the flow rate, the number of cells and the concentration of the cells in the sample.

 

PRODUCT QUESTIONS

Q1. What is the difference between SIM30 and SIM60?  

A1. SIM30 is suitable for analysing cells of

2µm - 12µm in size and

SIM60 is suitable for analysing cells of 6µm - 25µm. 

SIMs are interchangeable - so you don't have to buy a whole new Inish Analyser for each cell size range; just swap out the SIM as shown.

SIM30and60.png
Battery Indication

Q2. Does the Inish Analyser come with both the SIM30 and SIM60?  

A2. No.  The Inish Analyser comes with one SIM: SIM30 OR SIM60.  If you purchase Inish Analyser with SIM60, you can buy SIM30 separately and vice versa.

 

Q3. How often do I have to change the cartridge / microfluidic chip in the SIM?  

A3. This usually needs to be changed once per year. All Inish Analysers are shipped with a SIM box for annual return of the SIM for servicing.  The first service is free and includes a replacement cartridge / microfluidic chip; and recalibration and testing of the SIM.

Q4. What is the battery life of the Inish Analyser?

A4. 4 hours on full charge.

Q5. How do I know when to charge the Inish Analyser?

A5. There is a battery indicator the left-hand side of the device as shown.

Q6. Is the Inish Analyser CE-marked?

A6. Yes.

Q7. Is the Inish Analyser GMP compliant?

A7. No.

SOFTWARE QUESTIONS

Gating / Analysis of Populations

Q1. What do the results show?  

A1. Cell Counting & Viability results show a Flow Cytometry Standard (FCS) scatterplot where:

  • Live” cell population is represented by the population of green dots.

  • Dead” cell population is represented by the population of red dots.

  • Debris population is represented by the population of dots on the left-hand side of the scatterplot along the Y-axis.

Results are also displayed in a table, showing concentration (Total cells/mL) and viability (%).

Cell Counting Results - T cells

Transfection Efficiency results show a Flow Cytometry Standard (FCS) scatterplot where:

  • Live – Membrane closed” cell population is represented by the green dots.

  • Live - Membrane open” cell population is represented by the blue dots.

  • Dead” - cell population is represented by the red dots.

  • Debris population is represented by the dots on the left-hand side of the scatterplot along the Y-axis.

Results are also displayed in a table, showing concentration (Total cells/mL); Membrane open (%); Membrane closed (%) and Transfection Efficiency - predicted (%).

trans-6.png

Q2. Do the separating lines (i.e. horizontal/vertical gating lines) change with cell type?  Are the live/dead gate settings automatic?

A2. No.  The Inish Analyser includes a set of configuration files from which can be called-up to provisionally set the gates (i.e. move the horizontal/vertical lines to the correct position).  These may still be adjusted manually if necessary.

Q3. What do each axis of the graph represent? 

A3. X-axis:  Amplitude - gives us information about the size of the cells.

Y-axis:  Phase - gives us information about the membrane integrity of the cells.

Q4. What is the number of events you need to collect to analyse the data?

A4. Minimum 1,000 events.

Maximum 10,000 events.

Sorting, Exporting & Deleting Files

Q1. What is the storage memory of the Inish Analyser? / How many files can Inish Analyser store?  

A1. 32GB -  the Inish Analyser can store up to 25,000 files. 

Typically each file is ~1MB.

Q2. What is the format of the output file?

A2. FCS (Flow Cytometry Standard) or CSV.

Q3. We don't use pen/USB drive - how else can we export/transfer data?

A3. Use LAN (Local Area Network) to transfer files to another device.

Lan port

Q4. Do we need separate software to print results?

A4. No.  Results can be exported and printed as normal via your PC.

Q5. Can you delete files from the Inish Analyser?

A5. No.

General Software Queries

Q1. Do I have to log-in every time I use the machine?  

A1. Yes.

Q2. Is there a limit to the number of users you can create?

A2. No.

Q3. Can you enter Sample ID and other info from a computer instead? 

A3. Yes.  Contact Cellix for more information.

 
 
 
 

SAMPLE & WASTE TUBES

Q1. Is it safe to label the sample & waste tubes?  

A1. Yes.

Q2. How often do you have to change the waste tube?  

A2. After every assay.  If you don't change it and the tube over-fills, liquid will go into the pressure line causing damage to the instrument.

Q3. What is the size of the sample and waste tubes?  

A3. Volume of the sample tube:  500µL

Volume of the waste tube:  500µL

Sample & waste tubes are available from Cellix.

Section 5, Q3.png
 
 

WASH STEPS

Q1. Do I have to run the wash step after every assay?  

A1. No, but you must run the Wash step when going through the cleaning protocol:

  1. Before running any samples:

    • Wash with 360µL filtered deoinized water

    • Wash with 360µL Inish Buffer Solution

  2. After running all your samples:

    • Backflush with 360µL Inish Cleaning Fluid

    • Wash with 360µL Inish Cleaning Fluid

    • Wash with 360µL filtered deoinized water

Q2. What is the difference between "Wash" and "Backflush"?  

A2 Wash moves liquid (Inish Cleaning Fluid, Deionized water or Inish Buffer Solution) from left to right, while Backflush moves liquid (Inish Cleaning Fluid or Inish Buffer Solution) from right to left, as shown in the videos below.

Wash Step

Backflush

Q3. When do I need to run a wash?  

A3. You must run the Wash step when going through the cleaning protocol:

  1. Before running any samples:

    • Wash with 360µL filtered deoinized water

    • Wash with 360µL Inish Buffer Solution

  2. After running all your samples:

    • Backflush with 360µL Inish Cleaning Fluid

    • Wash with 360µL Inish Cleaning Fluid

    • Wash with 360µL filtered deoinized water

​​

Q4. When do I need to run a Backflush?  

A4. You must run the Backflush step in the following cases:

  • It is compulsory to run a Backflush after every Transfection Efficiency Assay.

  • After running all your samples: Backflush with 360µL Inish Cleaning Fluid before conducting remaining Wash steps, see A3 above.

  • If you are instructed to run a Backflush step via a Warning message.  You may receive a Warning message to Backflush the microfluidic impedance chip because it has become blocked.

Q5. How many washes can I run?  

A5. As many as you like, but one wash is usually sufficient. 

Q6. Is one wash enough for the same cell type?  

A6. Yes.

Q7. Does the wash solution need to be procured from Cellix?  

A7. Yes.  It is called Inish Cleaning Fluid.

RUNNING AN ASSAY

Q1. What is the recommended concentration of the cells for an assay?

A1. 50,000 - 2,000,000 cells/mL

Q2. What is the dilution factor for the cell sample?  

A2. 1:10. The standard protocol recommends 40µL of cell sample at the recommended concentration mixed with 360µL of Inish Analyser Buffer.

Q3. How much sample is required for each assay?  

A3. 40µL of cell sample at the recommended concentration is required for each assay.

App Note Infographic-5.png

Q4. Does the cell sample have to be homogeneous?  

A4. Ideally, yes.  Before adding the cell sample to the sample tube, pipette the sample up & down to ensure it it well mixed.  After adding the buffer to the cell sample, pipette up and down again to ensure it is well mixed.  

Q5. Does the assay stop automatically?  

A5. Yes.

 

MAINTENANCE & TROUBLESHOOTING

Q1. Do I have to run the wash step after every assay?  

A1. No, but you must run the Wash step when going through the cleaning protocol:

  1. Before running any samples:

    • Wash with 360µL filtered deoinized water

    • Wash with 360µL Inish Buffer Solution

  2. After running all your samples:

    • Backflush with 360µL Inish Cleaning Fluid

    • Wash with 360µL Inish Cleaning Fluid

    • Wash with 360µL filtered deoinized water

Q2. What is the difference between "Wash" and "Backflush"?  

A2 Wash moves liquid (Inish Cleaning Fluid, Deionized water or Inish Buffer Solution) from left to right, while Backflush moves liquid (Inish Cleaning Fluid or Inish Buffer Solution) from right to left, as shown in the videos below.

Wash Step

Backflush

Q3. When do I need to run a wash?  

A3. You must run the Wash step when going through the cleaning protocol:

  1. Before running any samples:

    • Wash with 360µL filtered deoinized water

    • Wash with 360µL Inish Buffer Solution

  2. After running all your samples:

    • Backflush with 360µL Inish Cleaning Fluid

    • Wash with 360µL Inish Cleaning Fluid

    • Wash with 360µL filtered deoinized water

​​

Q4. When do I need to run a Backflush?  

A4. You must run the Backflush step in the following cases:

  • It is compulsory to run a Backflush after every Transfection Efficiency Assay.

  • After running all your samples: Backflush with 360µL Inish Cleaning Fluid before conducting remaining Wash steps, see A3 above.

  • If you are instructed to run a Backflush step via a Warning message.  You may receive a Warning message to Backflush the microfluidic impedance chip because it has become blocked.

Q5. What can cause the chip to get blocked?

A5. Improper use of the Inish Analyser can cause the chip to become blocked such as:

  • Using a cell sample that has cell clumps or aggregates.

  • Not washing the Inish Analyser as recommended.

  • Not performing backflushes on the Inish Analyser as recommended.

Q6. Can I use ethanol for cleaning the Inish instead of Inish Cleaning Fluid?

A6. No - ethanol will damage the microfluidic impedance chip.  Under no circumstances should you use ethanol for wash steps or backflushes.

Q7. What if there are bubbles?  What should I do?

A7. Perform a backflush and repeat the wash steps.

Q8. What should I clean the outside of the Inish Analyser with?

A8. Any mild detergent is sufficient.

Q9. Is any system performance (i.e. calibration) needed before running an experiment?

A9. It is not necessary to perform a calibration assay before running an experiment.  However, we do recommend that the calibration assay be performed regularly, particularly if the Inish is in high use.

Q10. Can you briefly explain the calibration process of the Inish?

A10. The Inish Calibration Kit contains a bottle of beads of known concentration with a bottle of diluent.  The Calibration Assay runs the diluted bead sample through the Inish three times (3x).  The Inish automatically calculates the CV% of the three runs and gives a Pass/Fail result.

  • Pass result indicates that the Inish Analyser has detected the correct number of beads in the known concentration of the bead sample.

  • Fail result indicates that the Inish Analyser has detected an incorrect number of beads in the known concentration of the bead sample.

Inish Analyser Calibration Kit (1).jpg
Inish Calibration Kit Infographic.png