I NEED SOME HELP...

Mylen Pombo Tanda infomed!ICT.SLD.CU!MYLEN at GN.APC.ORG
Wed Jul 5 11:39:48 EST 1995


Havana City, July 6th 1995

TO ALL PERSONS WITH EXPERIENCE IN THE FIELD OF IMMUNE SYSTEM
RELATED DISORDERS:

Enclosed you will find copy of our kit for evaluating lymphoid
cells subpopulations by light microscopy. I would highly appreciate
your comments on components, procedure and possibilities of
applications. All suggestions for improving performance will be
very well received. 

     KIT FOR EVALUATION OF LYMPHOID CELLS SUBPOPULATIONS USING    
     LIGHT MICROSCOPY.

KIT Components: (for 60 tests).

*mAb panel: 
-CD3 mAb ................................1.2 mL
-CD4 mAb ................................1.2 mL
-CD8 mAb ..........,,,,,,,,,,,,,,,,,,,,,,1.2 mL

*Sheep anti mouse immunoglobulins/AP......4.8 mL

*Rabbit anti sheep immunoglobulins/AP.....4 8 mL

*Chromogenic substrate for alkaline phophatase:
-Naphtol As-Ax............................1 mg
-Levamisole...............................50  L
-Fast Red TR.............................5X1 mg

*Counterstaining solution:
-Methyl Green............................4.8 mL

*Adhesion printed glass slides 
(12 rings, 5 mm diameter)...................20

Procedure:
3 patients and 3 control samples/slide

Collect peripheral blood in heparinized medium. Isolate mononuclear
cells by centrifugation in Ficoll-Isopaque or a similar separation
medium. Count cells and adjust cell concentration to 106 cells per
mL. Apply 20  L cell suspension to slide wells.

1.Incubate with 20  L of mAb during 20 min. at r.t.
2.Rinse in TBS for 10 min.
3.Incubate with 20  L alkaline phosphatase conjugated sheep anti
mouse immunoglobulins for 20 min. at r.t.
4.Rinse in TBS for 5 min.
5.Incubate with 20  L alkaline phosphatase conjugated rabbit anti
sheep immunoglobulins during 20 min at r.t.
6.Rinse in TBS for 5 min.


7.Apply 20  L of Fast Red TR chromogen substrate solution for
alkaline phosphatase to the areas to be stained. Incubate slides
for 5-10 min at r.t.
8.Terminate the reaction by rinsing slides in distilled water.
9.Fix cells by placing slides in a koplin jar containing at the
bottom a cotton soaked with 37 % formaldehyde during 10 min.
10.Rinse with tap water.
11.Counterstain by applying 20  L Methyl Green to the samples.
Incubate at 37 oC during 10 min.
12.Rinse midly with tap water.
13.Air dry samples and mount with glicerol jelly.


14.Read under light microscope using 40X lens or 100X immersion
lens. Count a minimum of 200 cells. Estimate % positive cells using
the following formula:

% positive cells= No. positive cells  X 100 %
                    Total No. cells

Estimation of absolute CD4 T cell count can be performed through a
combination of the % CD4+ T cells calculated by the above formula
and hematological parameters.

Solutions:

*Tris buffer saline (TBS) pH 7,6
Tris (2-amino-2(hydroxymethyl)propane-1-3-diol)......60.55 g
NaCl.................................................85.20 g
Dissolve Tris and NaCl in approximately 500 mL of distilled water.,
Afjust pH to 7.6 with 1M ClH. Adjust volume to 1000 mL with
distilled water. Store at r.t. Dilute 1:10 before use. 

*0.1 M Tris buffer pH 8.2
Tris (2-amino-2(hydroxymethyl)propane-1-3-diol)......1.21 g
Dissolve Tris in 80 mL distilled water. Adjust volume to 100 mL
with distilled water. Store at 4 oC.

*Chromogenic Substrate Solution
Naphtol AS-MX sodium salt.....................1 mg
Levamisole (1 mol/L).........................50  L
Fast Red TR...................................1 mg
Disolve Naphtol AS-MX in Tris buffer. Add 50  L of levamisole.
Store 1 mL alicouts of the solution frozen.
Immediately before use, thaw an alicuot of Naphtol Tris buffer
solution. Add contents to 1 mg vial of Fast Red TR and dissolve
well. Filter the solution.  


   
                    





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