skip to main content, skip to Quick links, or skip to Search

main content

Contact

Ms. Sarah Bianco, Manager
Phone: 919.513.6248
Email: seschuet@ncsu.edu

Instrumentation

Policies

Flow Cytometry and Cell Sorting Laboratory

Blood Prep Requirements

Procedure for flow cytometry prep for blood:

  1. Dilute antibodies and prepare sample tubes with appropriate amount of antibodies in each tube. This is usually 100 uls of diluted antibody per antibody per tube. It is possible to mix directly conjugated antibodies in the tube together for staining.
  2. Make sure to prepare at LEAST ONE CELLS ONLY TUBE and one or more negative control tubes based on how many different isotypes of antibodies used and single color staining tubes if this is the first time these antibodies have been run by this flow operator. Consult with the individual flow operator before setting up experiment to see what they prefer.
  3. Collect blood in appropriate anticoagulant. You will need 100 uls of blood suspension per tube for each specimen. Make sure to run CBCs if total cell numbers are needed for interpretation of flow data. Spin the blood at sufficient speed to pellet cells(usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins). Remove plasma and save. Add PBS of Hanks Balanced Salt Solution in sufficient quantity to bring the blood back to its original volume.
  4. Add 100 uls of blood suspension to each sample tube.
  5. Place tubes on a rotating shaker on moderately fast shake, at room temp, in the dark for 20-30 mins. If there are questions about your cell surface markers, incubate antibodies and blood on ice on shaker. If incubating for longer periods, place on ice or at 40 C in dark. Blood & antibody tubes make be refrigerated overnight. Cover and keep in the dark. Overnight incubations should also have 2% fetal calf serum added to them.
  6. Wash each tube with 3-4 ml PBS and spin to pellet cells (usually about 1500 rpm on most standard sized lab centrifuges for 5-10 mins). Carefully pour off supernatant PBS(until red cells come just to the rim of the tube) or enough not to loose any pelleted cells.
  7. Resuspend cells by racking on plastic rack in resuidual liquid.