General guidelines for sorting bacteria with fluorescence-activated cell sorting (FACS)

This is not supposed to be a broad guide on how to use a FACS machine, since there is plenty of readily available information made by people who use these tools to study big cells. Instead, this will cover some techniques that will help you work with bacteria.

General guide for flow cytometry and bacteria

Gating bacteria and minimizing noise - This varies from machine to machine, but side-scatter can be used to resolve differences between cells when working with cells that are small. Forward scatter typically does not offer the same kind of resolution.

• Set threshold on SSC and adjust voltages for SSC and FCS. On an SSC-A vs FCS-A plot (log on both scales), you should see a population when running sample containing bacteria once your voltages and threshold are set right.
• Now, run a blank sample containing only buffer. Adjust voltages/threshold to limit any noise: 100-1000 events per second of noise is acceptable and should not significantly affect your measurements if you keep them out of your population gate. Iterate between this blank sample and the sample containing cells until you have found the right parameters that limits noise while allowing you to get 3,000-10,000 events per second when running a sample with cells.
• Once you have the ideal parameters, finish gating. Gate your population of cells on SSC-A vs FCS-A into a second plot with FCS-H vs FSC-A (both on log again). Gate singlets.

Optimizing sample preparation to avoid swarming

1. Prepare a spreadsheet with your dilution series and empty cells for events per second data.

Running samples to determine linear range - Make sure you have set up your parameters to identify cells and limit noise before running your dilution series and recording the events per second rates

1. At the FACS machine, run your buffer-only sample. Record the events per second on your spreadsheet. This is the background events per second.
2. Now, run each of your dilutions, recording the approximate events per second on the spreadsheet. Maintain the same speed and parameters throughout this.
3. Once you have finished your series, create a graph on excel to see the linear range. At high cell densities (10x dilution), it should dip or flatten out.
4. Pick one of the dilutions that remains in the linear range while still having plenty of cells to work with.

General guide for sorting bacteria

• Using bacteria with an antibiotic resistance marker (plasmid or genomic insertion) is advisable, because it is easy to get contamination when sorting cells. Especially if you are performing an evolution experiment that involves sorting, growing, and sorting iteratively.
• Make sure to sort into tubes made of polypropylene. Droplets stick on polystyrene and may not enter the media. These should contain media (saline, LB, or whatever) for your cells to directly fly into. Running through a small tube is probably very stressful (especially twice, see enrichment sorts below), so having some nutrient in the media will help your sorted cells remain viable.
• The nozzle size shouldn't matter so much, as long as your cells are not swarming. A 100μm (common size) nozzle will work fine as long as you do not sort too fast.
• When actively sorting, aim for ~5k events total events per second.
• Some machines allow you to change the sorting stringency. This is advisable. For example, the Sony MA900 has an ultra purity mode that rejects ~20-50% of droplets which do not meet the sorting criteria. This can be adjusted to purity or semi-purity mode, or yield mode if purity is not important.

Enrichment sorts - If your sorts are not working well, you can perform a 2-step sort to achieve sorts with higher 'purity'.

1. Sort a large amount of cells from your population of interest with no rejects at a high rate into an empty tube. Aim for 10-100x the final number of cells you want to sort.
2. Once you have finished sorting the first time, load the SORTED cells (now further diluted in sheath fluid) and begin sorting again, this time with a high stringency. You may have to bump up the speed rate due to the dilution caused by sheath.
   • The 2nd sort should go into a tube with media.

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General guidelines for sorting bacteria with fluorescence-activated cell sorting (FACS)

This is not supposed to be a broad guide on how to use a FACS machine, since there is plenty of readily available information made by people who use these tools to study big cells. Instead, this will cover some techniques that will help you work with bacteria.

General guide for flow cytometry and bacteria

• Set threshold on SSC and adjust voltages for SSC and FCS. On an SSC-A vs FCS-A plot (log on both scales), you should see a population when running sample containing bacteria once your voltages and threshold are set right.

• Once you have the ideal parameters, finish gating. Gate your population of cells on SSC-A vs FCS-A into a second plot with FCS-H vs FSC-A (both on log again). Gate singlets.

Optimizing sample preparation to avoid swarming

Performing dilution series

1. If you are growing cells in an autofluorescent media like LB, you will need to wash the cells. Pellet 1mL of stationary culture for at 5k rcf for 5 minutes, decant LB with a pipette tip and resuspend with 1mL of your buffer. If you are growing cells in minimal media like M9 (even with casamino acids), you do not need to wash your cells and can directly add them to your dilution series.
2. Prepare tubes with different volumes of your buffer and make the dilution series of your samples. Begin at a dilution of 10x (adding 100uL of culture to 900uL of buffer), and finish at ~1000x or more. You will have to perform serial dilutions to get a good range. In my experience, a 100x dilution seems to work OK when working with E.coli grown in M9 (0.4% glucose) or ADP1 grown in LB. Include a tube with no bacteria added to know what the background events per second are for your dilution.
3. Prepare a spreadsheet with your dilution series and empty cells for events per second data.

Running samples to determine linear range - Make sure you have set up your parameters to identify cells and limit noise before running your dilution series and recording the events per second rates

1. At the FACS machine, run your buffer-only sample. Record the events per second on your spreadsheet. This is the background events per second.

1. Once you have finished your series, create a graph on excel to see the linear range. At high cell densities (10x dilution), it should dip or flatten out.

General guide for sorting bacteria

• Using bacteria with an antibiotic resistance marker (plasmid or genomic insertion) is advisable, because it is easy to get contamination when sorting cells. Especially if you are performing an evolution experiment that involves sorting, growing, and sorting iteratively.
• Make sure to sort into tubes made of polypropylene. Droplets stick on polystyrene and may not enter the media. These should contain media (saline, LB, or whatever) for your cells to directly fly into. Running through a small tube is probably very stressful (especially twice, see enrichment sorts below), so having some nutrient in the media will help your sorted cells remain viable.
• The nozzle size shouldn't matter so much, as long as your cells are not swarming. A 100μm (common size) nozzle will work fine as long as you do not sort too fast.
• When actively sorting, aim for ~5k events total events per second.
• Some machines allow you to change the sorting stringency. This is advisable. For example, the Sony MA900 has an ultra purity mode that rejects ~20-50% of droplets which do not meet the sorting criteria. This can be adjusted to purity or semi-purity mode, or yield mode if purity is not important.

Enrichment sorts - If your sorts are not working well, you can perform a 2-step sort to achieve sorts with higher 'purity'.

1. Sort a large amount of cells from your population of interest with no rejects at a high rate into an empty tube. Aim for 10-100x the final number of cells you want to sort.
2. Once you have finished sorting the first time, load the SORTED cells (now further diluted in sheath fluid) and begin sorting again, this time with a high stringency. You may have to bump up the speed rate due to the dilution caused by sheath.
   • The 2nd sort should go into a tube with media.

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General guidelines for sorting bacteria with fluorescence-activated cell sorting (FACS)

This is not supposed to be a broad guide on how to use a FACS machine, since there is plenty of readily available information made by people who use these tools to study big cells. Instead, this will cover some techniques that will help you work with bacteria.

General guide for flow cytometry and bacteria

Gating samples - This varies from machine to machine, but side-scatter can be used to resolve differences between cells when working with cells that are small. Forward scatter typically does not offer the same kind of resolution.

• Set threshold on SSC and adjust voltages for SSC and FCS. On an SSC-A vs FCS-A plot (log on both scales), you should see a population when running sample containing bacteria once your voltages and threshold are set right.
• Now, run a blank sample containing only buffer. You should see a negligible amount of noise (~100 events per second). Adjust voltages/threshold to limit any noise. Iterate between your blank sample and the sample containing buffer until you have found the right parameters that limits noise while allowing you to get 3,000-10,000 events per second when running a sample with cells.
• Once you have the ideal parameters, finish gating. Gate your population of cells on SSC-A vs FCS-A into a second plot with FCS-H vs FSC-A (both on log again). Gate singlets.

Optimizing sample preparation to avoid swarming

Performing dilution series

1. If you are growing cells in an autofluorescent media like LB, you will need to wash the cells. Pellet 1mL of stationary culture for at 5k rcf for 5 minutes, decant LB with a pipette tip and resuspend with 1mL of your buffer. If you are growing cells in minimal media like M9 (even with casamino acids), you do not need to wash your cells and can directly add them to your dilution series.
2. Prepare tubes with different volumes of your buffer and make the dilution series of your samples. Begin at a dilution of 10x (adding 100uL of culture to 900uL of buffer), and finish at ~1000x or more. You will have to perform serial dilutions to get a good range. In my experience, a 100x dilution seems to work OK when working with E.coli grown in M9 (0.4% glucose) or ADP1 grown in LB. Include a tube with no bacteria added to know what the background events per second are for your dilution.
3. Prepare a spreadsheet with your dilution series and empty cells for events per second data.

Running samples to determine linear range - Make sure you have set up your parameters to identify cells and limit noise before running your dilution series and recording the events per second rates

1. At the FACS machine, run your buffer-only sample. Record the events per second on your spreadsheet. This is the background events per second.
2. Now, run each of your dilutions, recording the approximate events per second on the spreadsheet.
3. Once you have finished your series, create a graph on excel to see the linear range. At high cell densities (10x dilution), it should dip or flatten out.
4. From this range, pick one of the dilutions that remains in linear range while still having plenty of cells to work with.

General guide for sorting bacteria

• Using bacteria with an antibiotic resistance marker (plasmid or genomic insertion) is advisable, because it is easy to get contamination when sorting cells. Especially if you are performing an evolution experiment that involves sorting, growing, and sorting iteratively.
• Make sure to sort into tubes made of polypropylene. Droplets stick on polystyrene and may not enter the media. These should contain media (saline, LB, or whatever) for your cells to directly fly into. Running through a small tube is probably very stressful (especially twice, see enrichment sorts below), so having some nutrient in the media will help your sorted cells remain viable.

• When actively sorting, aim for ~5k events total events per second.
• Some machines allow you to change the sorting stringency. This is advisable. For example, the Sony MA900 has an ultra purity mode that rejects ~20-50% of droplets which do not meet the sorting criteria. This can be adjusted to purity or semi-purity mode, or yield mode if purity is not important.

Enrichment sorts - If your sorts are not working well, you can perform a 2-step sort to achieve sorts with higher 'purity'.

1. Sort a large amount of cells from your population of interest with no rejects at a high rate into an empty tube. Aim for 10-100x the final number of cells you want to sort.
2. Once you have finished sorting the first time, load the SORTED cells (now further diluted in sheath fluid) and begin sorting again, this time with a high stringency. You may have to bump up the speed rate due to the dilution caused by sheath.
   • The 2nd sort should go into a tube with media.

Comments

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General guidelines for sorting bacteria with fluorescence-activated cell sorting (FACS)

This is not supposed to be a broad guide on how to use a FACS machine, since there is plenty of readily available information made by people who use these tools to study big cells. Instead, this will cover some techniques that will help you work with bacteria.

General guide for flow cytometry and bacteria

Gating samples - This varies from machine to machine, but side-scatter can be used to resolve differences between cells when working with cells that are small. Forward scatter typically does not offer the same kind of resolution.

• Set threshold on SSC and adjust voltages for SSC and FCS. On an SSC-A vs FCS-A plot (log on both scales), you should see a population when running sample containing bacteria once your voltages and threshold are set right.
• Now, run a blank sample containing only buffer. You should see a negligible amount of noise (~100 events per second). Adjust voltages/threshold to limit any noise. Iterate between your blank sample and the sample containing buffer until you have found the right parameters that limits noise while allowing you to get 3,000-10,000 events per second when running a sample with cells.
• Once you have the ideal parameters, finish gating. Gate your population of cells on SSC-A vs FCS-A into a second plot with FCS-H vs FSC-A (both on log again). Gate singlets.

Optimizing sample preparation to avoid swarming

Performing dilution series

1. If you are growing cells in an autofluorescent media like LB, you will need to wash the cells. Pellet 1mL of stationary culture for at 5k rcf for 5 minutes, decant LB with a pipette tip and resuspend with 1mL of your buffer. If you are growing cells in minimal media like M9 (even with casamino acids), you do not need to wash your cells and can directly add them to your dilution series.
2. Prepare tubes with different volumes of your buffer and make the dilution series of your samples. Begin at a dilution of 10x (adding 100uL of culture to 900uL of buffer), and finish at ~1000x or more. You will have to perform serial dilutions to get a good range. In my experience, a 100x dilution seems to work OK when working with E.coli grown in M9 (0.4% glucose) or ADP1 grown in LB. Include a tube with no bacteria added to know what the background events per second are for your dilution.
3. Prepare a spreadsheet with your dilution series and empty cells for events per second data.

Running samples to determine linear range - Make sure you have set up your parameters to identify cells and limit noise before running your dilution series and recording the events per second rates

1. At the FACS machine, run your buffer-only sample. Record the events per second on your spreadsheet. This is the background events per second.
2. Now, run each of your dilutions, recording the approximate events per second on the spreadsheet.
3. Once you have finished your series, create a graph on excel to see the linear range. At high cell densities (10x dilution), it should dip or flatten out.
4. From this range, pick one of the dilutions that remains in linear range while still having plenty of cells to work with.

General guide for sorting bacteria

• Using bacteria with an antibiotic resistance marker (plasmid or genomic insertion) is advisable, because it is easy to get contamination when sorting cells. Especially if you are performing an evolution experiment that involves sorting, growing, and sorting iteratively.
• Make sure to sort into tubes made of polypropylene. Droplets stick on polystyrene and may not enter the media. These should contain media (saline, LB, or whatever) for your cells to directly fly into. Running through a small tube is probably very stressful (especially twice, see enrichment sorts below), so having some nutrient in the media will help your sorted cells remain viable.
• The nozzle size shouldn't matter so much, as long as your cells are not swarming. A 100μm (universal size for most FACS machines) nozzle will work fine as long as you do not sort too fast.
• When actively sorting, aim for ~5k events total events per second.
• Some machines allow you to change the sorting stringency. This is advisable. For example, the Sony MA900 has an ultra purity mode that rejects ~20-50% of droplets which do not meet the sorting criteria. This can be adjusted to purity or semi-purity mode, or yield mode if purity is not important.

Enrichment sorts - If your sorts are not working well, you can perform a 2-step sort to achieve sorts with higher 'purity'.

1. Sort a large amount of cells from your population of interest with no rejects at a high rate into an empty tube. Aim for 10-100x the final number of cells you want to sort.
2. Once you have finished sorting the first time, load the SORTED cells (now further diluted in sheath fluid) and begin sorting again, this time with a high stringency. You may have to bump up the speed rate due to the dilution caused by sheath.
   • The 2nd sort should go into a tube with media.

Comments

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