This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
The following guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the flow cytometry (FACS) experiments. We at Boster Bio are committed to helping our customers “get better results”. While the troubleshooting guide below covers a multitude of problems encountered while performing flow cytometry experiments, we do not expect it to be the exclusive solution to any problems during your specific experiment. We do hope that you will find the information beneficial to you and useful as a reference guide in troubleshooting any FACS problems you may encounter. If you ever need more assistance with your flow cytometry experiments, please contact the Boster Support Team by email at [email protected]
Download troubleshootingnhandbooks for IHC, Western blot and ELISA for FREE.
Troubleshooting guidesS.No. | Possible Cause | Solution |
---|---|---|
1 | The antibodies are degraded or expired |
|
2 | The fluorescence of the fluorochrome has faded |
|
3 | The antibody concentration is too low for detection |
|
4 | Expression of target antigen is too low |
|
5 | Antigen-antibody binding is sub-optimal |
|
6 | The intracellular antigen is not accessible |
|
7 | The intracellular antigen is getting secreted |
|
8 | The surface antigen is getting internalized |
|
9 | The fluorescence on stained cells has bleached |
|
10 | A low expressing antigen has been paired with a dim fluorochrome |
|
11 | The primary and secondary antibodies are not compatible |
|
12 | The laser and PMT settings are not compatible with fluorochrome or PMT voltage is too low for the fluorescent specific channel |
|
13 | The fluorescent signal is over compensated |
|
S.No. | Possible Cause | Solution |
---|---|---|
1 | The antibody concentration is too high |
|
2 | Unbound antibodies are trapped in the cells in the case of intracellular staining |
|
3 | A high expressing antigen is paired with bright fluorochrome |
|
4 | The PMT voltage is too high for the fluorescent specific channel |
|
5 | The fluorescent signal is under-compensated |
|
6 | Inadequate blocking |
|
S.No. | Possible Cause | Solution |
---|---|---|
1 | Excess, unbound antibodies are present in the sample |
|
2 | Non-specific cells are targeted |
|
3 | High auto-fluorescence |
|
4 | Presence of dead cells |
|
S.No. | Possible Cause | Solution |
---|---|---|
1 | The cells are lysed or damaged |
|
2 | Bacterial contamination |
|
3 | Incorrect instrument settings for scatter |
|
4 | Presence of dead cells |
|
5 | Presence of un-lysed RBCs (red blood cells) |
|
S.No. | Possible Cause | Solution |
---|---|---|
1 | Event rate is low due to low cell number |
|
2 | Event rate is low due to sample clumping |
|
3 | Incorrect instrument settings |
|
4 | No events due to a clogged sample injection tube |
|
5 | Event rate is too high due to concentrated sample |
|
6 | Event rate is too high due to air in flow cells and/or sheath filter |
|
S.No. | Possible Cause | Solution |
---|---|---|
1 | Excessive paraformaldehyde |
|
2 | Sample was not kept on ice |
|
3 | Sample fixed too long |
|
Here are the 300 most popular antibodies.
Flow cytometry is a popular cell biology technique that utilizes laser-based technology to count, sort, and profile cells in a heterogeneous fluid mixture. FACS is an abbreviation for fluorescence-activated cell sorting, which is a flow cytometry technique that further adds a degree of functionality.
See MoreBoster Bio protocols for flow cytometry offer a step-by-step overview of the procedure. Use this guide as a primer or a quick reference guide, and see our product datasheets or sample preparation guides for more details.
See MoreBinding of antibody to surface antigen can stimulate the cells and alter the expression of intracellular signalling proteins. Saponin does not alter the surface antigen epitopes so surface staining can be done afterwards. Methanol is compatible with...
See MoreGet some of the best Bosterbio's flow cytometry optimization tips. This guide includes protocols, optimization tips, troubleshooting tips, and more on flow cytometry.
See MoreEvery flow cytometry (or FACS) experiment begins with sample preparation. Check out our flow cytometry sample preparation guide to learn how to prepare your samples for flow cytometric analysis.
See MoreAntibody titration is recommended to determine the correct concentration of an antibody for the optimum signal. Test different dilutions of the antibody to zero in on the lowest concentration that gives the strongest signal in positive control...
See MoreFlow Cytometry Flow Cytometry Sample Preparation Optimization Sample preparation and cell quality tips Whenever possible, use freshly isolated cells rather than frozen and thawed cells. To increase viability of thawed cells, perform the initial...
See MoreIsotype control Cells incubated with isotype control antibodies (antibodies usually raised against an antigen that should not be present in your cells). Secondary antibody control Cells incubated only with the secondary antibody. Viability control...
See More