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Department of Microbiology and Immunology: Flow Cytometry Core Facility
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  • Protocols and Reagents
    • Immunofluorescence Staining for Immunotyping
    • Cell Cycle Staining using PI
    • Sorting and Staining Buffers
    • Lysing red blood cells
  • Sample Submission
    • Appointments and Pricing
    • Sample Preparation
  • Instruments
    • BD FACS Symphony- 16 Color Self Service Cytometer
    • BD LSRFortessa- 16 color cytometer
    • Cytek Aurora CS-cell sorter
    • BD FACS Celesta-12 color self-service cytometer
    • Cytek Northern Lights-Full Spectrum Cytometer
    • Hematology Analyzer-VETSCAN HM5
  • Data Analysis and Citation
  1. University of Arkansas for Medical Sciences
  2. College of Medicine
  3. Department of Microbiology and Immunology
  4. Research Cores
  5. Flow Cytometry Core Facility
  6. Protocols and Reagents
  7. Sorting and Staining Buffers

Sorting and Staining Buffers

Sorting Buffer

  • PBS or Hanks (Ca/Mg ++ free)
  • 25mM Hepes pH= 7.0
  • 0.5-2% FBS (heat inactivated)
  • 1mM EDTA.
  • 0.2 mm sterile filtered

Notes:

Culture Media is not good as a sort buffer because outside the confines of the incubator, the pH tends to become basic, which may be the reason for low viability and recovery. The addition of Hepes will help to stabilize the pH.

For cells that don’t stick together, you can modify the sort buffer to omit the EDTA. You should use the least amount of FBS the cells need to remain happy.

The addition of EDTA will help reduce the stickiness of some cell types. The concentration of EDTA should not exceed 5mM.

Staining Buffer

Place on ice or store at 4C until use.  You can make up 1 L at a time and store at 4C, as long as it is kept sterile for staining cells.

To make 1L:

2g BSA

2ml 0.5mM EDTA

0.1% sodium azide

1x PBS to 1L

Filter and sterilize if needed.

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