Preparation of TonsilLysate

Purpose

The protocol describes how to prepare human tonsil lysate for use in purification of ICAM-1,LFA-1,and PNAd.

Materials

Safety Equipments

Lab Coat

Latex Gloves

Face Mask

Bench Paper

Fresh human tonsil tissue

Parafilm

Scissors

Stainless steel mesh screen (200 mesh=74 mm,Type 316; Tylinter,Mentor,Ohio)

RPMI-1640 medium

Triton X-100

Lysis Buffer:

50 mM Tris-HCl,pH 7.5

150 mM NaCl

0.02% NaN3

10 mg/ml Aprotinin

10 mg/ml Pepstatin

10 mg/ml Leupeptin

1 mM PMSF (Made fresh the day of experiment.Dissolve in 100% EtOH)

1 mM Benzamidine

5 mM Iodoacetamide

Whatman Paper

Centrifuge Bottles

Funnel

Squeeze bottle

Several Beakers

Homogenizer (Fisher PowerGen 125)

50ml Corning tubes

Ice Bucket & Ice

Dounce homogenizer (40ml Pyrex Cat No.7727-40)

1 liter bottle

Stir bar

2 Side-arm flasks

Procedure

All work must be done at 4℃.

Collect 50 grams of tonsil tissue.(this should yield a large amount of ICAM-1,LFA-1,and PNAd)

Note 1: Collecting this much tonsils can take some time.So it is good to collect from more than one source.

Note 2: You can prep each sample as you receive it or wait until you have a ~ 50g of tonsils.

If you decide to prep them right away then perform steps 2 to 4 and then freeze at -80℃.

If you want to wait until you have ~50g of tonsil,you should immediately freeze tonsils at -80℃.

Clear a work area in the 4℃ walk-in fridge.Lay down bench paper and assemble all you materials.

Mince the tonsils with the scissors.Try and make the pieces a small as possible.

Note: You can use a small glass plate as a cutting surface to make the job easier.

Fold the wire mesh in the shape of a cone and place in a funnel.Place funnel on the mouth of the beaker,to collect RPMI-1640.

Pour RPMI-1640 into a squeeze bottle.Wash the minces tonsils with copious amounts of RPMI-1640.Wash a small amount of tonsils at a time.

(At this point you can freeze the tonsils until you have enough)

Set up the homogenizer with the large probe (1cm diameter).

Put a small amount of minced tonsil in the 50ml Corning tube and add lysis buffer to ~10ml mark.

ｉｎｓｅｒｔ probe to the tube and the cover the neck of the tube and the main body of the probe with parafilm.This is very important.This prevents aerosols from escaping during the homogenizing process.

Homogenize in small burst to avoid heating the sample.

Note 1: Homogenize till you liquefy the sample.You may still see a little bit of white strands,but these are connective tissue and will not completely break down.

Note 2 : The probe will get clogged with connective tissue.Clean it of with tissue paper and rinse in with water by pulsing the probe to remove the rest of the stuck connective tissue.Once clean again you can continue homogenizing your sample.

Collect the homogenized tissue in a beaker that is kept on ice.

Using the dounce homogenizer,homogenize the collected sample using an up & down and left & right hand motion.Do this at least 10 times to get a very fine sample.

Note: To avoid spilling you sample,don’t use more than the volume stated on the side of the dounce homogenizer.

When you are done transfer homogenized tissue to a 1 liter bottle and add in the stir bar.

Bring the volume up to 1 liter by adding more of the Lysis buffer.

Add Triton-X 100 to a final concentration of 2% by volume.

Note: The Triton-X-100 will not dissolve immediately.It will go into solution slowly over night.

Stir gently over night in the 4℃ walk-in.

Transfer the liquid to centrifuge bottles and spin down lysate at 8000g for 1 hour.

Decant lysate in a clean beaker.Discard pellet.

Filter lysate twice,using Buckner Funnel and Whatman paper as follows:

Cut the Whatman paper to cover the bottom of the Buckner funnel.

Place funnel on side-arm flask and attach.

Attach the first flask to the second and to the vacuum.

Filter the lysate through the Whatman,changing it often as it gets clogged.

Filtrate may now be stored a 4℃ until it is time to run over an affinity column.

Note: Don't store for more than a couple of days at 4℃.

References

K.D.Puri,E.B.Finger,G.Gaudernack,and T.A.Springer (1995).Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules.J Cell Biol 131:261-270.