Preparation of Tonsil Lysate

Purpse

The prtcl describes hw t prepare human tnsil lysate fr use in purificatin f ICAM-1,LFA-1,and PNAd.

Materials

Safety Equipments

Lab Cat

Latex Glves

Face Mask

Bench Paper

Fresh human tnsil tissue

Parafilm

Scissrs

Stainless steel mesh screen (200 mesh=74 mm,Type 316; Tylinter,Mentr,hi)

RPMI-1640 medium

Tritn X-100

Lysis Buffer:

50 mM Tris-HCl,pH 7.5

150 mM NaCl

0.02% NaN3

10 mg/ml Aprtinin

10 mg/ml Pepstatin

10 mg/ml Leupeptin

1 mM PMSF (Made fresh the day f experiment.Disslve in 100% EtH)

1 mM Benzamidine

5 mM Idacetamide

Whatman Paper

Centrifuge Bttles

Funnel

Squeeze bttle

Several Beakers

Hmgenizer (Fisher PwerGen 125)

50ml Crning tubes

Ice Bucket & Ice

Dunce hmgenizer (40ml Pyrex Cat N.7727-40)

1 liter bttle

Stir bar

2 Side-arm flasks

Prcedure

All wrk must be dne at 4℃.

1.Cllect 50 grams f tnsil tissue.(this shuld yield a large amunt f ICAM-1,LFA-1,and PNAd)

Nte 1: Cllecting this much tnsils can take sme time.S it is gd t cllect frm mre than ne surce.

Nte 2: Yu can prep each sample as yu receive it r wait until yu have a ~ 50g f tnsils.

If yu decide t prep them right away then perfrm steps 2 t 4 and then freeze at -80℃.

If yu want t wait until yu have ~50g f tnsil,yu shuld immediately freeze tnsils at -80℃.

2.Clear a wrk area in the 4℃ walk-in fridge.Lay dwn bench paper and assemble all yu materials.

3.Mince the tnsils with the scissrs.Try and make the pieces a small as pssible.

Nte: Yu can use a small glass plate as a cutting surface t make the jb easier.

4.Fld the wire mesh in the shape f a cne and place in a funnel.Place funnel n the muth f the beaker,t cllect RPMI-1640.

5.Pur RPMI-1640 int a squeeze bttle.Wash the minces tnsils with cpius amunts f RPMI-1640.Wash a small amunt f tnsils at a time.

(At this pint yu can freeze the tnsils until yu have enugh)

6.Set up the hmgenizer with the large prbe (1cm diameter).

7.Put a small amunt f minced tnsil in the 50ml Crning tube and add lysis buffer t ~10ml mark.

8.ｉｎｓｅｒｔ prbe t the tube and the cver the neck f the tube and the main bdy f the prbe with parafilm.This is very imprtant.This prevents aersls frm escaping during the hmgenizing prcess.

9.Hmgenize in small burst t avid heating the sample.

Nte 1: Hmgenize till yu liquefy the sample.Yu may still see a little bit f white strands,but these are cnnective tissue and will nt cmpletely break dwn.

Nte 2 : The prbe will get clgged with cnnective tissue.Clean it f with tissue paper and rinse in with water by pulsing the prbe t remve the rest f the stuck cnnective tissue.nce clean again yu can cntinue hmgenizing yur sample.

10.Cllect the hmgenized tissue in a beaker that is kept n ice.

11.Using the dunce hmgenizer,hmgenize the cllected sample using an up & dwn and left & right hand mtin.D this at least 10 times t get a very fine sample.

Nte: T avid spilling yu sample,dn’t use mre than the vlume stated n the side f the dunce hmgenizer.

12.When yu are dne transfer hmgenized tissue t a 1 liter bttle and add in the stir bar.

13.Bring the vlume up t 1 liter by adding mre f the Lysis buffer.

14.Add Tritn-X100 t a final cncentratin f 2% by vlume.

Nte: The Tritn-X-100 will nt disslve immediately.It will g int slutin slwly ver night.

15.Stir gently ver night in the 4℃ walk-in.

16.Transfer the liquid t centrifuge bttles and spin dwn lysate at 8000g fr 1 hur.

17.Decant lysate in a clean beaker.Discard pellet.

18.Filter lysate twice,using Buckner Funnel and Whatman paper as fllws:

Cut the Whatman paper t cver the bttm f the Buckner funnel.

Place funnel n side-arm flask and attach.

Attach the first flask t the secnd and t the vacuum.

Filter the lysate thrugh the Whatman,changing it ften as it gets clgged.

19.Filtrate may nw be stred a 4℃ until it is time t run ver an affinity clumn.

Nte: Dn't stre fr mre than a cuple f days at 4℃.

References

K.D.Puri,E.B.Finger,G.Gaudernack,and T.A.Springer (1995).Sialmucin CD34 is the majr L-selectin ligand in human tnsil high endthelial venules.J Cell Bil 131:261-270.