Monitor the lower limit fluorescence sensitivity to be sure your instrument is ready for immunophenotyping assays.
BioSure® Negative Control (osmium fixed chicken red blood cells) is a negative standard for assessing the lower limit of fluorescence sensitivity of a flow cytometer. The fixation process quenches cellular fluorescence and results in a particle that has very low autofluorescence. Daily use of this standard in immunophenotyping assays can show that the flow cytometer is capable of detecting particles with lower fluorescence than unstained target cells. Discrimination of dimly fluorescent target cells from the negative control cells proves that the instrument is sensitive enough to perform the immunophenotyping assay.
The Negative Control is prepared from chicken erythrocytes that have been processed to remove leukocytes. The fixed cells are suspended in Dulbecco's PBS and supplied in 2.0 mL volumes. The cell count per milliliter is at least 2.5 X 107 negative cells.
The Negative Control is stable at 2° - 8° C until the expiration date denoted on each vial label. Freshly diluted (1:10) preparations are stable for at least 12 hours when stored at 2° - 8° C.
PREPARATION OF WORKING SOLUTION
The recommended working solution is a 1:10 dilution of the stock solution in particulate-free physiological saline or isotonic buffered saline. This provides an adequate cell density and sufficient working solution for multiple assays.
Typical Negative Control graphs
1. Thoroughly mix the Negative Control stock, and add one or two drops into a test tube (13 X 75 mm).
2. Add 0.9 mL of diluent, mix, and evaluate.
Although the Negative Control has been thoroughly washed to remove residual fixative, the chemical is hazardous, and it is recommended that gloves and eye protection be worn when handling this product.
- Initial Standardization
- Daily Monitoring
- Sensitivity Assessment
- Alignment & Linearity
- Biological Safety Data
- Material Safety Data Sheet
Know the Lower Limit of Fluorescence with BioSure® Negative Control
This procedure is used to assess a flow cytometer's lower limit of fluorescence by using the Negative Control, which consists of chicken erythrocytes that have been fixed in osmium tetroxide (OsO4). The fixation process quenches cellular fluorescence and results in a particle that has very low autofluorescence. Daily use of this negative standard in immunophenotyping assays demonstrates that the flow cytometer is capable of detecting particles with lower fluorescence than unstained target cells. Discrimination of dimly fluorescing target cells (cellular autofluorescence or isotypic control cells) from the negative control cells proves that the instrument is sensitive enough to perform the immunophenotyping assay. This becomes especially critical when measuring markers of low antigen density on the cell surface such as with activation, natural killer cell or some B cell antibodies.
Fluorescence Sensitivity Procedure
- 1. Align the instrumentation optics, if necessary.
- 2. Prepare a cell suspension of leukocytes from a venous blood sample collected from a control donor. Lyse the erythrocytes using the same lysing method which will be used on the laboratory samples. Fix the lysed whole blood sample in a 2% buffered paraformaldehyde solution.
- 3. Place the fixed sample on the cytometer. Set the instrument parameters as follows: FSC-linear, SSC-linear, FL1-log, FL2-log. Adjust the FSC amplifier gain to position the left edge of the lymphocyte population below mid-scale on the forward scatter display. Adjust the SSC to position the top of the granulocyte population close to the edge of the side scatter display. Record the FSC and SSC amplifier and gain settings.
- 4. Set a gate around the lymphocytes and activate the gate. Change the display parameters to FL1 and FL2.
- 5. Adjust the high voltage on the Fluorescence 1 PMT to position the unstained lymphocytes in the first decade of the FL1 display. Adjust the high voltage on the Fluorescence 2 PMT to position the unstained lymphocytes in the first decade of the FL2 display. Repeat the PMT adjustment for any other parameters (FL3 for example), if desired. Record the PMT voltage settings and the median channel numbers for the populations in each of the fluorescence parameters.
- 6. Repeat Steps 1 - 5 at least nine times using venous blood collected from different control donors. Average the PMT settings for each of the parameters - SSC, FL1 and FL2. Record the average setting and median channel number for each of the parameters. Since the FSC detector is a photo diode, only the amplifier setting will be recorded.
- 7. Prepare a fresh dilution of the Negative Control to achieve a flow rate of about 500 - 1000 events per second. Place the tube on the cytometer.
- 8. Adjust the FSC amplifier gain until both peaks of the negative control population can be seen in the forward scatter histogram display. Once the gain has been established, this setting will be used as the FSC reference target value and should not be altered in subsequent daily monitorings. Record the FSC gain setting.
- 9. Proceed to an FL1 vs FL2 dot plot or bit mapped display. Keep the same fluorescence PMT settings in steps 5 and 6. Determine and record the median channel number for each of the fluorescence parameters.
The median fluorescence channel number of the Negative Control should be lower than the median fluorescence channel number for the unstained lymphocytes. Autofluorescence of unstained lymphocytes has a fluorescence equivalent intensity of approximately 1000 molecules of fluorescein (excitation at 488 nm). Since the cells in the Negative Control have little or no autofluorescence, a difference in fluorescence between the two populations will demonstrate that the flow cytometer is sensitive enough to distinguish between unstained cells and dimly fluorescing cells.
If the median fluorescence channel number of the Negative Control is equal to or greater than the median fluorescence channel number of the unstained lymphocytes, then instrument sensitivity in the fluorescence parameters may be suspect. Cleaning, realignment or servicing of the instrument may be necessary to restore sensitivity in the fluorescence parameters.
Biological Safety Data
This product is manufactured by BioSure®, Inc. as a biological research product. To the
best of our knowledge, this product does not contain concentrations of carcinogenic,
teratogenic or chemical agents that exceed 0.1% or greater in accordance with OSHA’s
Hazardous Communication Standard 29CFR1910.1200 (d) (5) (i-iv). This product is
exempt from the Material Safety Data Sheet requirements. Biological research controls
should be handled using proper laboratory safety procedures, such as gloves, eye
protection etc. Disposal can be by normal sewage. However, autoclaving or incineration
is recommended should this product be mixed with hazardous or infectious materials
such as human or animal serum, plasma, whole blood or tissue. If you have further
questions, please call us at 800-345-2267 or email us at