Author: Unkown. Date: 7/30/01
SODIUM AZIDE: highly toxic. May cause heritable genetic damage. Very toxic by inhalation, in contact with skin, and if swallowed. heating may cause explosion. Avoid contact with metals. Do not breath dust. Do not get solutions in eyes, on skin, or on clothing.
FITC, Phycoeurithrin,TRI-color, and BIOTIN: small quantities of conjugated chemicals are present in the antibody preparations. Avoid contact with eyes, skin and clothing.
Lab coat
gloves
Wear a dust mask when working with dry sodium azide
drain
Use either 96-well plates, or Falcon 2025 tubes
FACs Buffer:
á 1X PBS + 1% FCS + 0.02% azide
á 500ml 1XPBS, add 5ml FCS and 5ml of 2% azide
ACK buffer: prepare according to gold book protocol
1. Add 1ml non-sterile ACK lysis buffer to each blood sample. Mix by pipetting gently up and down. Incubate at room temp. for approx. 10 min.
2. Spin in the microcentrifuge for 3-4 min. @ 3 - 4,000 x g.
3. Aspirate supernatant carefully. Lymphocytes should sit at bottom, with platelets next. DonŐt try to get all the platelets.
4. Add 200ul x number of stains (1 or 2) for FACS buffer to each tube.
5. Resuspend cell pellet and transfer 200ul of the cells to each well of a 96 well round bottomed assay plate. (If 400ul is added, 200ul goes into each well.) Keep track on template (lid).
6. Spin the plate at 1000 rpm for 5 min in refrigerated table-top centrifuge.
7. * During spin, prepare 1st stage antibodies:
FACS buffer volume=50ul x (number of samples + 1)
to this, add appropriate amount of first stage antibodies (volume buffer/dilution factor)
8. FLICK plate right away when spin comes down.
9. Add 50ul of each first stage antibody. Resuspend the cells by gentle pipetting.
10. Incubate for 30 minutes on ice and covered. (at least 20min.)
11. Wash by adding 150ul FACs buffer and spin at 1000 rpm for 5 min.
12. Wash again (for bio primary staining) by adding 200 ul FACs buffer and spinning at 1000 rpm for 5 min. * Otherwise skip to 18.
13. During this wash, prepare 2nd stage antibodies:
FACs buffer volume=50ul x (number of samples + 1)
to this add appropriate amount of antibody
FLICK plate right away when spin comes down.
14. Add 50ul second antibody. Resuspend the cells by gentle pipetting.
15. Incubate for 30 minutes on ice. Keep covered.
16. Wash by adding 150ul FACs buffer and spinning at 1000 rpm for 5 min.
17. FLICK plate right away when spin comes down.
18. Add 200ul FACs buffer. Cells are now ready to read.
á If you need to FIX the cells (i.e. canŐt get FACs time within 4 hours of preparing blood) then after step 15 do the following:
19. Add 200ul FACs buffer. Resuspend the cells by gentle pipetting.
20. Spin plate at 1000 rpm, 5 min.
21. FLICK pate right away when spin comes down.
22. Add 200ul FACs FIX,. Resuspend the cells by gentle pipetting.
23. Wrap the plate in tinfoil. Keep in refrigerator until ready to read.