Applications for indirect immunofluorescence technique
Reagents and Equipment:
Conjugate, GMo17F: FITC-conjugated Goat anti-Mouse immunoglobulin antiserum,solid phase adsorbed with human immunoglobulins,(cat#RDI-M1204clb) Remove aggregates by centrifugation at 1000 x g for 10 minutes.Dilute as mentioned in the procedure.
Wash-and dilution buffer for mononuclear cells, Phosphate Buffer Saline, containing 0.2% BSA (w/v) (PBS/BSA).
Wash-and dilution buffer for platelets, Sequestrine buffer (Seg),storage 1 month at 2-8'C.
10x stock solution, dissolve in 1 Liter distilled water.
Na2,HPO4,2H2O: 15.65g
Na2EDTA-2H20: 16.65g (Complexon)
NaCI: 450g
Prior to use dilute in distilled water, add BSA till final concentration of 0.2% (w/v), Mix and adjust pH to 6.8
Permeabilization buffer, (BFA).
Buffered Formaldehyde Acetone, storage 1 month at 2-8'C. Dissolve in 450 ml distilled water:
Na2HPO4 2H-O: 300mg
KH2PO4: 1500mg
Add to 30 ml of this buffer 50 ml acetone and 25 ml formaldehyde, mix and adjust pH to 7.5,
Fixation buffer, PFA/BSA:
Para-Formaldehyde 1% in PBS,containing 0.2% BSA (pH7.2).
Lysing solution, NH4 C1:
NH4CL: 0.155M / KHCO3 / Na2EDTA 0.1 mM (pH 7.4).
Embedding medium:
70% glycerol in PBS.
Microwell plates and tubes:
Microwell plated (96 wells,V bottom) or plastic flowcytometry tubes.
Cell isolation:
1. Isolation of mononuclear cells.
1.1 Draw blood into a blood collecting tube containing EDTA, store at room temperature (18-25'C).
1.2 Isolate the mononuclear cells, preferably within 4 hours after collection,by standard density gradient centrifugation
(e.g. Ficoll-Paque,Pharmacia).
1.3 Wash cells at least twice with PBS/BSA and adjust cell concentration to 1 x 10 7 cells/ml in PBS/BSA.
2. Isolation of platelets.
2.1 Draw blood into a collecting tube containing EDTA, (Care should be taken to prevent activation of platelets.The
expression of most platelet activation markers is maximal after stimulation with 1 U/ml thrombin).
2.2 Prepare a Platelet Rich Plasma (PRP) by centrifugation of EDTA-anticoaguated blood at 500 x g for 5 minutes, use no brake,transfer supernatant (PRP) with a plastic pipette into a 50 ml conical plastic centrifuge tube.
2.3 Wash by filling the tube with Seq,mix and centrifuge at 1600 x g for 10 minutes.Discard the supernatant and repeat once more. If erythrocytes are visible in the pellet after the first washing,lyse the erythrocytes by adding 5ml NH4C1 for 10 minutes in an ice-water bath, then continue with the last wash.
2.4 Adjust the platelet concentration to 1 x 10 e08 (100 MILLION) cells/ml in Seq.
Procedures:
1. Mononuclear cell membrane flow cytometry /microscopy.
1.1 Transfer 45 ul of the mononuclear cell suspension into microwell plate or tubes and add 5 ul monoclonal antibody*, mix gently and incubate for 30 minutes at 2-8'C.
1.2 Wash by mixing and adding PBS/BSA to the microwell plate
(1st wash 150 ul, 2nd wash 200 ul) or tubes (2 ml), centrifuge at 500 x g for 5 minutes and aspirate the supernatant. Repeat this procedure once more.
1.3 Add 50 ul GoMo17F,diluted 1:80 in PBS/BSA. Mix and incubate for 30 minutes at 2-8'C in the dark.
1.4 Wash: see step 1.2.
1.5 Prepare cells for analysis:
For flow cytometry resuspend the cells by adding 200 ul PFA/BSA to the microwell plates or tubes. If a microwell plate was used, the contents are transferred to appropriate tubes.For fluorescence microscopy,resuspend the cells in 50 ul embedding medium,transfer cells to a microscope slide and place a cover glass.
2. Mononuclear cell cytoplasmic flow cytomery (1).
2.1 For 20 tests, resuspend 1x10 E07 (10 million) mononuclear cells in 250 ul of BFA and incubate for only 2 seconds at 18-25'C.(This incubation will stop by the addition of the wash buffer,a longer BFA fixation will diminish the fluorescence intensity).
2.2 The cells are immediately washed by mixing and adding PBS (2-8'C). Centrifuge at 500 x g for 5 minutes, repeat this procedure once more. Use 5 x 10 E05 (100 thousand) cells per test.
2.3 Transfer 45 ul of the cell suspension into microwell plate or tubes and add 5 ul monoclonal antibody*.
Mix gently and incubate for 30 minutes at 2-8'C.
2.4 Wash by mixing and adding PBS/BSA to the microwell plate
(1st wash 150 ul, 2nd wash 200 ul) or tubes (2 ml), centrifuge at 500 x g for 5 minutes and aspirate the supernatant, repeat this procedure once more.
2.5 Add 50 ul GoMo17F, diluted 1:80 in PBS/BSA. Mix and incubate for 30 minutes at 2-8'C in the dark.
2.6 Wash: see step 2.2.
2.7 For analysis, the cells are resuspended in 200 ul PBS/BSA,if a microwell plate was used, the contents are transferred to appropriate tubes.
3. Mononuclear cell cytomplasmic fluorescence microscopy.
3.1 Adjust cell concentration to 2 x 10 E06 (2 million) cells/ml.
3.2 Prepare slides by briefly centrifuging 50 ul PBS/BSA at maximum speed in a cytocentrifuge,next centrifuge 50 ul cell suspension for 5 minutes at 500 g.
3.3 Allow the slides to dry for 30 minutes at 18-25'C.
3.4 Fix the cells in a slide jar containing acetone for 10 minutes at 18-25'C.
3.5 Wash the slides in a slide jar containing PBS under contin uous gentle agitation for 3 separate 5 minutes changes.
Carefully dry the slides around the spot.
3.6 Dispense 50 ul diluted monoclonal antibody* over the spot. Incubate for 30 minutes at 18-25'C in a humid dark container
3.7 Wash the slides: see step 3.5.
3.8 Dispense 50 ul GoMo17F, diluted 1:160 in PBS,over the spot and incubate for 30 minutes at 18-25'C in a humid dark container.
3.9 Wash the slides: see step 3.5.
3.10 Dispense one drop of embedding medium over the spot and place a cover glass.
4. Platelet membrane flow cytometry/microscopy.
4.1 Transfer 45 ul platelet suspension into the microwell plate or tubes and add 5 ul monoclonal antibody*.Mix gently and incubate for 30 minutes at 2-8'C.
4.2 Wash by mixing and adding Seq to the microwell plate (1st wash 150 ul, 2nd wash 200 ul) or tubes (2 ml). Centrifuge at 1000 x g for 5 minutes and aspirate the supernatant,repeat this procedure once more.
4.3 Add 50 ul GoMo17F, diluted 1:80 in Seq. Mix and incubate for 30 minutes at 2-8'C in the dark.
4.4 Wash: see step 4.2
4.5 Prepare cells for analysis:
For flow cytometry, resuspend the cells by adding 200 ul Seq to the microwell plate or tubes. If a microwell plate was used the contents are transferred to appropriate tubes. For fluoescence microscopy, resuspend the cells in 50 ul embedding medium, transfer cells to a microscope slide and place a cover glass.
NOTE:
In general, 5 ul undiluted monoclonal antibody can be used. Alternatively an optimal dilution can be determined.To determine background fluorescence always use a negative control from the same isotype.
REFERENCE:
1. Slaper-Cortenbach, I.C.M. et al.,The flow-cytometric detection of Terminal deoxynucleotidyl Transferase (TdT)and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias,Blood,72 (1988).