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]

Troubleshooting Guides

Download troubleshootingnhandbooks for IHC, Western blot and ELISA for FREE.

troubleshooting_box_image Troubleshooting guides
  • Weak Fluorescence Intensity or No Fluorescent Signal

    S.No. Possible Cause Solution
    1 The antibodies are degraded or expired
    • Ensure that antibodies are stored as per the instructions of manufacturer.
    • Keep track of antibody stocks; make sure products are not expired.
    • If titrating antibodies and storing aliquots of the same, add sodium azide in the storage buffer at 0.09%.
    2 The fluorescence of the fluorochrome has faded
    • Be sure to store the conjugated antibodies away from light exposure. Fresh antibodies will be needed.
    3 The antibody concentration is too low for detection
    • Titrate the antibodies before use to find the optimal amount to use for your specific experiment.
    • Use negative (unstained) and positive controls.
    4 Expression of target antigen is too low
    • Check literature for antigen expression in different cell types and use a suitable positive control.
    • Whenever possible, use freshly isolated cells as opposed to frozen samples.
    • Optimize cell culture/stimulation protocols in case the antigen (e.g. cytokines) expression depends on prior in vitro treatment.
    5 Antigen-antibody binding is sub-optimal
    • Check the species specificity of the antibody.
    • Optimize the antibody incubation time and temperature.
    • Consider using biotinylated primary antibodies and an additional biotin-steptavidin step to amplify the signal.
    6 The intracellular antigen is not accessible
    • Optimize cell permeabilization protocols.
    7 The intracellular antigen is getting secreted
    • Target must be membrane bound or cytoplasmic to be detected. Try using a Golgi blocker such as Brefeldin A.
    8 The surface antigen is getting internalized
    • Perform all protocol steps at 4°C and use ice cold reagents. We recommend you permeablize on ice.
    9 The fluorescence on stained cells has bleached
    • Acquire the cells immediately after staining.
    • Add a fixative (like PFA) to the samples if storing for an extended duration; alcohol fixatives are best avoided.
    10 A low expressing antigen has been paired with a dim fluorochrome
    • Always pair the weak antigens with bright fluorochromes such as PE or APC.
    11 The primary and secondary antibodies are not compatible
    • Use a secondary antibody that was raised against the species in which the primary antibody was raised.
    12 The laser and PMT settings are not compatible with fluorochrome or PMT voltage is too low for the fluorescent specific channel
    • Ensure that proper instrument settings are loaded prior to acquisition.
    • Use suitable positive and negative controls to optimize settings for every fluorochrome.
    13 The fluorescent signal is over compensated
    • Use MFI alignment instead of visual comparison to compensate.
  • Saturated or Excess Fluorescent Signal

    S.No. Possible Cause Solution
    1 The antibody concentration is too high
    • Titrate the antibodies before use to find the optimal amount to use for your specific experiment.
    • Use appropriate positive and negative controls.
    2 Unbound antibodies are trapped in the cells in the case of intracellular staining
    • Wash the cells adequately after each antibody incubation step and include Tween or Triton X in wash buffers.
    3 A high expressing antigen is paired with bright fluorochrome
    • Always pair strong antigens with dimmer fluorochromes such as FITC or Pacific Blue.
    4 The PMT voltage is too high for the fluorescent specific channel
    • Ensure proper instrument settings are loaded prior to acquisition.
    • Use suitable positive and negative controls to optimize settings for every fluorochrome.
    5 The fluorescent signal is under-compensated
    • Use MFI alignment instead of visual comparison to compensate.
    6 Inadequate blocking
    • Add 1% to 3% blocking agent with antibody as well as a blocking step (dilute antibodies in blocking solution), and increase the blocking time.
  • High Background or Non-specific Staining

    S.No. Possible Cause Solution
    1 Excess, unbound antibodies are present in the sample
    • Wash cells adequately after every antibody incubation step
    2 Non-specific cells are targeted
    • Include an isotype control to subtract any Fc binding signal.
    • Block the Fc receptors on cells with Fc blockers, BSA or FBS prior to antibody incubation.
    • Include additional washing steps.
    • Include a secondary antibody-conjugate control to subtract any non-specific signal from the conjugate.
    • Select a secondary antibody that does not cross react.
    3 High auto-fluorescence
    • Always include an unstained control to subtract the auto-fluorescence signal.
    • For cells with naturally high auto-fluorescence (e.g. neutrophils), use fluorochromes that emit in the red channel where auto-fluorescence is minimal (e.g. APC).
    • If the above solution is not possible, use very bright fluorochromes for these cell types to amplify the signal above the auto-fluorescence level.
    • Avoid storing the cells in a fixative solution for long durations; analyse cells soon after staining if possible.
    4 Presence of dead cells
    • Always sieve the cells once before acquiring and sorting to remove any dead cell debris.
    • Include viability dyes like PI or 7-AAD to gate out any dead cells.
    • Use freshly isolated cells as opposed to frozen cells whenever possible.
  • High Background Scatter or Abnormal Scatter Profile of Cells

    S.No. Possible Cause Solution
    1 The cells are lysed or damaged
    • Optimize sample preparation to avoid cell lysis.
    • If possible, do not vortex or centrifuge cells at high speeds.
    • Use fresh buffers.
    • Avoid storing the stained cells for long durations.
    2 Bacterial contamination
    • Store stained cells and antibodies properly to avoid bacterial growth. Bacteria will exhibit low levels of auto fluorescence.
    • Practice proper sterile cell culture techniques to prevent bacterial contamination.
    3 Incorrect instrument settings for scatter
    • Ensure proper instrument settings are loaded prior to acquisition.
    • Use fresh, healthy cells to correctly set the FSC and SSC settings for that cell type.
    4 Presence of dead cells
    • Always sieve the cells once before acquiring and sorting to remove any dead cell debris.
    • Use freshly isolated cells as opposed to frozen cells whenever possible.
    5 Presence of un-lysed RBCs (red blood cells)
    • Ensure RBC cells lysis is complete – check using a microscope.
    • Use fresh RBC lysis buffer.
    • Wash as many times as needed to remove RBC debris.
    • If using PBMCs after a ficoll gradient, optimize the procedure to minimize RBC contamination in the lymphocyte interphase.
  • Abnormal Event Rate

    S.No. Possible Cause Solution
    1 Event rate is low due to low cell number
    • Keep the minimum cell count at 1X106/ml.
    • Ensure the cells are mixed well with gentle pipetting.
    2 Event rate is low due to sample clumping
    • Always sieve the cells once prior to acquiring and sorting to remove debris.
    • Ensure the cells are mixed well with gentle pipetting before staining, and again before running your samples.
    3 Incorrect instrument settings
    • Adjust the threshold parameter as needed.
    4 No events due to a clogged sample injection tube
    • Unclog flow cytometer injection tube as per instrument manufacturer's instructions (typically run 10% bleach for 5-10 min, followed by dH2O for 5-10 min).
    5 Event rate is too high due to concentrated sample
    • Dilute the cell count to 1X106/ml.
    6 Event rate is too high due to air in flow cells and/or sheath filter
    • Refer to the instrument manual for the appropriate steps.
  • Loss of Epitope

    S.No. Possible Cause Solution
    1 Excessive paraformaldehyde
    • Paraformaldehye can release methanol in its breakdown, which may affect the staining. Make sure to use only 1% paraformaldehyde.
    2 Sample was not kept on ice
    • Keep antibodies at 4 °C to prevent loss of activity. This also prevents active phosphatases and proteases from altering the epitope of interest.
    3 Sample fixed too long
    • Optimize fixation protocol. Fixing the samples for too long may cause damage to cells. Most cells only need to be fixed for less than 15 minutes.

Popular Antibodies

Here are the 300 most popular antibodies.

acetylcholinesterase antibodyADAM10 antibodyADAMTS13 antibodyadiponectin antibody
AGO2 antibodyAHR antibodyALK antibodyalkaline phosphatase antibody
AMH antibodyANG antibodyannexin a1 antibodyAPOE antibody
AQP1 antibodyaquaporin 4 antibodyARC antibodyATF4 antibody
ATF6 antibodyATM antibodyATRX antibodyBAX antibody
bcl xl antibodyBCR antibodybeclin 1 antibodyBRCA1 antibody
CAD antibodycalreticulin antibodycaspase 1 antibodycaspase 8 antibody
catalase antibodycathepsin b antibodyCcl2 antibodyCCR4 antibody
CCR5 antibodycd11b antibodycd11c antibodyCd14 antibody
cd16 antibodyCD163 antibodyCD19 antibodycd2 antibody
cd20 antibodyCD200 antibodyCD24 antibodyCD27 antibody
cd30 antibodyCD33 antibodyCD34 antibodyCD36 antibody
Cd40 antibodyCD44 antibodycd45 antibodyCD47 antibody
CD63 antibodyCD68 antibodycd8 antibodyCD80 antibody
CD81 antibodyCD86 antibodyCd9 antibodyCDK2 antibody
CDK4 antibodyceruloplasmin antibodyCHAT antibodyclaudin 5 antibody
collagen i antibodycollagen ii antibodycreb antibodyCrp antibody
csf antibodyCSF1R antibodyCtla4 antibodyCXCL10 antibody
CXCR3 antibodyCXCR4 antibodycyclin antibodycytokeratin 5 antibody
desmoglein 3 antibodyDKK1 antibodyDLL4 antibodyDNMT1 antibody
doublecortin antibodyEEA1 antibodyEGFR antibodyenos antibody
EPCAM antibodyERBB2 antibodyerythropoietin antibodyEZH2 antibody
fak antibodyFAP antibodyFAS antibodyFGF21 antibody
Fgf23 antibodyFGFR1 antibodyFLT3 antibodyFOS antibody
FOXO1 antibodyFOXP3 antibodyGAL antibodyGAPDH antibody
GATA3 antibodyGDF15 antibodyglut4 antibodygp100 antibody
GPI antibodyGPX4 antibodygrp78 antibodyHGF antibody
HIF1A antibodyHLA-A antibodyhsp90 antibodyhuntingtin antibody
iba1 antibodyICAM1 antibodyIcos antibodyIDS antibody
IFNAR1 antibodyIgf1r antibodyIl10 antibodyIL13 antibody
IL15 antibodyIl17a antibodyIL18 antibodyIL1B antibody
IL2 antibodyIL33 antibodyIL5 antibodyIL6R antibody
IL8 antibodyinos antibodyIRF3 antibodyislet 1 antibody
JAK2 antibodyjnk antibodyKEAP1 antibodyKLF4 antibody
L1CAM antibodylactoferrin antibodylamin a antibodyLAMP1 antibody
LAT antibodyleptin antibodyLif antibodyLOX antibody
lysozyme antibodyMAG antibodyMAX antibodyMBP antibody
MDM2 antibodyMERTK antibodymesothelin antibodyMICA antibody
MIF antibodyMLH1 antibodyMMP2 antibodyMMP9 antibody
MOG antibodyMTOR antibodyMUC2 antibodymyeloperoxidase antibody
NANOG antibodynephrin antibodynestin antibodyNGF antibody
NLRP3 antibodyNOTCH1 antibodyNOX4 antibodyNPY antibody
NRP1 antibodyoccludin antibodyosteocalcin antibodyosteopontin antibody
p300 antibodyp63 antibodyparkin antibodyparp antibody
PAX6 antibodyPAX8 antibodyPCSK9 antibodypd l1 antibody
PDGFRA antibodyPDGFRB antibodyperilipin antibodyperk antibody
Pf4 antibodypgp9.5 antibodyPML antibodyppar gamma antibody
PROX1 antibodyPTEN antibodyrab7 antibodyRAC1 antibody
RAD51 antibodyrea antibodyRET antibodyrip3 antibody
RIPK1 antibodyRUNX2 antibodySHH antibodySIRT1 antibody
SMAD2 antibodysmad3 antibodySNAP25 antibodySOD1 antibody
SOD2 antibodysomatostatin antibodySOX10 antibodySOX2 antibody
SOX9 antibodySP1 antibodySPR antibodySRC antibody
STAT3 antibodySTAT6 antibodysurvivin antibodySYK antibody
TAZ antibodyTBK1 antibodytdt antibodyTERT antibody
thrombin antibodyThy1 antibodytim 3 antibodyTLR2 antibody
TLR3 antibodyTLR4 antibodyTnf antibodytopoisomerase i antibody
transferrin antibodytrkb antibodyTSG101 antibodyTSHR antibody
TSLP antibodyubiquitin antibodyVDAC1 antibodyvegf antibody
VWF antibodyXBP1 antibodyZEB2 antibody