Human Antibody Titration Protocol

Last Updated 11/19/99

Contacts: Steve De Rosa (derosa@fhcrc.org)

Materials

Qty

Location

Order Info

Staining Media

 

B-013 on Steve’s shelf in Fridge 6

 

Fixing Solution

 

B-011 over Steve’s bench

 

Antibody

 

B-011 Fridge 5

 

Paraformaldehyde

 

B-011 over Steve’s bench

 

FACS tubes

 

 

Falcon 35-2052 (no caps), 2058 (caps)

Large ice bucket

 

 

 

96-well plates

 

 

Flexible round-bottom disposable plates

Aluminum Plate Holders

 

B-011 over Tomy Centrifuge

 

 

 

Preliminary calculations:

1.      Determine the concentration of antibody in the stain (ug/ul).

2.      Calculate the volume required for 1-4 ug of antibody. This is the typical amount of antibody used to start a titration. eg. 4 ug antibody = 16 ul at 0.25 ug/ul.

 

Staining:

  1. For speed and convenience, samples can be stained in a 96 well plate.
  2. In the first well of each set, make the starting volume in the amount of staining media to total 100ul. (Ex. starting volume=20ul antibody: 40ul antibody, 60ul media)
  3. Add 50ul of staining media to the next nine wells for each antibody.
  4. Make 1:2 serial dilutions, taking 50ul. Discard the last 50ul.
  5. Spin enough cells so that you have a total of 0.5 million per sample.
  6. Resuspend cells to concentration of 0.5 million/50ul
  7. Add cells to stain dilutions. Sit for 15 minutes on ice.
  8. Wash by adding 150ul staining media
  9. Spin for 3 min @ 4oC at 2000rpm.
  10. Aspirate and wash in 200ul staining media
  11. Repeat spin
  12. Aspirate and wash in 200ul staining media
  13. Repeat spin
  14. Resuspend in 200ul 0.5%paraformaldehyde Transfer to FACS tubes arranged in a similar pattern to the plate (Note: If you use every other column of the plate, it is easy to dispense into adjacent tubes in a rack using a multi-channel pipetter. If you choose to use every column, you will want to arrange tubes in the rack by odd and even column numbers to facilitate transfer.)
  15. Happy sorting!

 

Recipes:

Staining media: deficient hRPMI

            3% NCS

            0.02% Azide (1/500 of 10% stock)

            optional: 1mM EDTA (mouse/clumpy cells only)

 

Fixing solution:  deficient hRPMI

0.5% paraformaldehyde (1:8 of 4% stock)

Paraformaldeyhde (4%)

Paraformaldehyde is very toxic and aerates easily. Avoid breathing in the powder. Use a fume hood if necessary.

 

1.      Mix required amount of paraformaldehyde (4g/100ml) to 2/3 final volume in ddH2O.

1.      Heat to 60C while stirring in a fume hood (monitor temperature with thermometer).

2.      Add 1 drop 2N NaOH to clear the solution.

3.      Remove heat and add 1/3 vol 3x PBS.

4.      Let cool and adjust to pH 7.2 with HCL.

5.      Filter.