Magni:Protocols/flowcy: Difference between revisions
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(New page: {{Magni}} <div style="padding: 10px; width: 720px; border: 5px solid #000000;"> = Preparation of samples for flow cytometry = *Grow cells under the desired conditions. *At the required ti...) |
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*At the required time points, dilute cells 5-fold in filter-sterilized PBS 1X supplemented with gentamycin (final concentration of 1 mg/ml) in order to stop translation. | *At the required time points, dilute cells 5-fold in filter-sterilized PBS 1X supplemented with gentamycin (final concentration of 1 mg/ml) in order to stop translation. | ||
*incubate the diluted cells under the same conditions as before for about 1 hour and then start analysis. | *incubate the diluted cells under the same conditions as before for about 1 hour and then start analysis. | ||
*At least 100,000 events should be collected and stored for each sample during analysis. | |||
*After analysis, data can be gated and analyzed by the FloMax (Partec) software (only available in the same computer of the machine). | |||
*Otherwise, acquired data files can be imported in MATLAB via the fca_readfcs script (Laszlo Balkay). The acquired log-binned values of forward scatter (FSC), side scatter (SSC) and fluorescence (FL1) are integers in the range 0-4095. Log-binned FSC, SSC and FL1 values can be linearized, gated and background-subtracted as required | |||
NOTES: | NOTES: | ||
*The available Partec PAS II flow cytometer is equipped with an argon ion laser using the 488 nm blue line for excitation and a 515-545 nm band-pass filter is used for emission. For this reason, only GFP can be detected. | *The available Partec PAS II flow cytometer is equipped with an argon ion laser using the 488 nm blue line for excitation and a 515-545 nm band-pass filter is used for emission. For this reason, only GFP can be detected. | ||
*At least 1 million of cells should be present in the final sample for analysis and the sample volume should be at least 1.5 ml. To do this, the 5-fold dilution can be adjusted as required. Cell count can be estimated by measuring the OD600 of culture and by using an OD600-CFU calibration curve (this is strain-dependent!!!). | *At least 1 million of cells should be present in the final sample for analysis and the sample volume should be at least 1.5 ml. To do this, the 5-fold dilution can be adjusted as required. Cell count can be estimated by measuring the OD600 of culture and by using an OD600-CFU calibration curve (this is strain-dependent!!!). | ||
</div> | </div> | ||
Latest revision as of 07:36, 5 May 2013
Preparation of samples for flow cytometry
- Grow cells under the desired conditions.
- At the required time points, dilute cells 5-fold in filter-sterilized PBS 1X supplemented with gentamycin (final concentration of 1 mg/ml) in order to stop translation.
- incubate the diluted cells under the same conditions as before for about 1 hour and then start analysis.
- At least 100,000 events should be collected and stored for each sample during analysis.
- After analysis, data can be gated and analyzed by the FloMax (Partec) software (only available in the same computer of the machine).
- Otherwise, acquired data files can be imported in MATLAB via the fca_readfcs script (Laszlo Balkay). The acquired log-binned values of forward scatter (FSC), side scatter (SSC) and fluorescence (FL1) are integers in the range 0-4095. Log-binned FSC, SSC and FL1 values can be linearized, gated and background-subtracted as required
NOTES:
- The available Partec PAS II flow cytometer is equipped with an argon ion laser using the 488 nm blue line for excitation and a 515-545 nm band-pass filter is used for emission. For this reason, only GFP can be detected.
- At least 1 million of cells should be present in the final sample for analysis and the sample volume should be at least 1.5 ml. To do this, the 5-fold dilution can be adjusted as required. Cell count can be estimated by measuring the OD600 of culture and by using an OD600-CFU calibration curve (this is strain-dependent!!!).
