Flow Cytometry Protocol

Required Materials

  • 10X Phosphate-buffered saline (PBS), pH 7.2 (Sigma, catalog # P5493)
  • Buffers
    • PBS - dilute 1 part of 10X PBS with 9 parts of nanopure H2O
    • Evident's Live Cell Blocking buffer (EF-XXX-LCB-0030)

Procedure

  1. Obtain cells of interest and wash twice with cold PBS via centrifugation for 10 minutes at 4°C.
    Note: It is suggested that centrifugation of live cells be performed in the range of 500-700g
  2. Aliquot 1 x 106 cells per sample or control to be analyzed into 1.5mL microcentrifuge tubes.
    Note: Cells may be counted using a hemacytometer or other instrument following manufacturer's protocol
  3. Centrifuge cells for 5 minutes to pellet. Discard the supernatant.
  4. Resuspend pellet in cold blocking buffer and incubate on ice for 20-30 minutes.
  5. Centrifuge cells to pellet. Discard the supernatant.
  6. Add primary antibody at manufacturer's recommended dilution in blocking buffer. Resuspend cells in 50µL of the diluted primary antibody solution and incubate on ice for 30min-1hour.
    Note: Primary antibody incubation times vary. Refer to manufacturer's recommendations.
  7. Centrifuge cells to pellet; discard supernatant. Wash cells twice with blocking buffer, centrifuging for 5 minutes to pellet. Discard the supernatant.
  8. Dilute EviFluor in blocking buffer to a final OD range of 0.005 to 0.01 at the peak absorption of the EviFluor.
    Example: If the peak absorption of your EviFluor sample is 0.3 OD, dilute 1:30 for a final OD of 0.01.
  9. Resuspend cells in 50µL of the diluted EviFluor. Incubate 1 hour on ice.
  10. Â Centrifuge cells to pellet; discard supernatant. Wash cells twice with PBS, centrifuging for 5 minutes. Discard the supernatant.
  11. Â Resuspend pellet in 400µL PBS and transfer to analysis tube.
  12. Â Analyze with flow cytometer. Refer to instrument manual for analysis instructions.

Troubleshooting

  1. If I have a mixed population of cells how do I separate out the cells I want to analyze?

    It is often possible to analyze one subpopulation of cells within a mixed population without physically separating out the population of interest simply by probing a marker that is highly specific to the population of interest. However, if this is not an option there are several methods available for separation of mixed populations of cells. It is the customer's responsibility to research this because there are many different types of cells and separation methods.
  2. How do I count my cells?

    There are many methods to count cells. Instrumentation that will automatically give you a value for cell concentration from a small sample of cells in solution may be available in your lab. Alternatively, a traditional method to count cells employs the use of a hemacytometer, a small tool that contains a grid structure to enable cell counting. You simply put a sample of your cells under a slide and count them using a microscope. Using a hemacytometer often requires the cells are first dyed with Trypan Blue (since the cells are transparent and thus hard to see without the dye). The Trypan Blue (Sigma, catalog # T8154 or ATCC catalog # 30-2402) comes with a protocol describing its use.
  3. How do I count my cells using the hemacytometer?

    Please refer to the hemacytometer manufacturer's recommended procedure.
  4. How do I know how fast my centrifuge goes in g force units when mine says rpm?

    Conversion charts or conversion equations facilitating the calculation of g force units from rpm are often available from instrument manufacturers. If you no longer have the instrument manual this information may be available online or obtained by contacting the instrument's vendor.
  5. Why do you recommend a dilution range for the EviFluors?

    The affinity of both the primary antibody for its target and the EviFluor for the primary antibody can vary. Therefore, it may be necessary to alter the amount of secondary required for sufficient staining of the cells.
  6. My primary antibody's manufacturer doesn't recommend a dilution for flow. How much should I add?

    A titration of the primary may be necessary to determine the optimal dilution. As a general rule, adding too much is better than adding too little because you can usually wash off the excess unbound antibody.
  7. I have nonspecific binding with the EviFluors. What should I do?

    We suggest you use our Live Cell Blocking Buffer. Also, you may be using the EviFluors at too high a concentration for your assay. Try increasing the dilution of the EviFluors. Furthermore, your primary antibody may be nonspecifically binding.
  8. I don't see any signal. What should I do?

    The primary antibody could be too dilute. Increase the concentration of the primary. Alternatively, you may increase the concentration of the EviFluors.
  9. Do I have to use your EviFluor Live Cell Blocking Buffer?

    Use of Evident's Live Cell Blocking Buffer is not a requirement for flow cytometry. However, the scientists at Evident have found this to be the most effective reagent for reduction of background binding and therefore its use is highly recommended.
  10. Why is it important to incubate at cold temperatures?

    The viability of cells is maintained better at cold temperatures (~4°C). Therefore, it is highly recommended that all incubations occur on ice and that all buffers are kept chilled throughout the flow cytometry procedure.

Order EviFluor Quantum Dot Conjugates and Blocking Buffers Below

Product Availability
Conjugate Wavelength Part Number Concentration Price
Goat anti-Mouse 520nm
600nm
620nm
680nm
 EF-C11-GMO-0XXX 2.5uM $289
Goat anti-Rabbit EF-C11-GRA-0XXX $289
Goat anti-Rat EF-C11-GRT-0XXX $289
Streptavidin EF-C11-STR-0XXX $439
Biotin EF-C11-BT1-0XXX $439
Specifications
Product Colors

Add to cart:

Product Availability
Part Number Description Volume Price
EF-XXX-LCB-0030 Live Cell Blocking Buffer 30mL $59
EF-XXX-FCB-0030 Fixed Cell Blocking Buffer 30mL $59
Specifications

Add to cart:

Questions?