White Blood Cell Cytospin
Materials:
Fresh blood
Histopaque 1.083 (Sigma)
0.5% EDTA (RNAse Free)
PBS/10% BSA (RNAse Free)
PBS/1% BSA (RNAse Free)
Statspin Cytofuge 2
Single chamber cytospin devices
Pre-cleaned glass slides (Baked for >2 hours at 100°C )
Staining Reagents: 75% Ethanol; 95% Ethanol.
Procedure:
Preparation of cell suspension
1. Aliquot 1.0ml of Histopaque into a 1.7 ml microcentrifuge tube.
2. Overlay 0.4 ml of fresh blood on top of the Histopaque
3. Centrifuge for 15 minutes at speed 3
4. Recover buffy coat at interface between plasma and histopaque
5. Centrifuge for 15 minutes at speed 3 and remove supernatant
6. Mix with .1ml .5% EDTA and .9 ml of PBS/10%BSA.
7. Centrifuge for 5 minutes at speed 3.
8. Remove supernatant, resuspend cells in .1ml of .5% EDTA and .9 ml of PBS/10%BSA.
Cytospin slide preparation
1. Assemble single chamber cytospin device according to Package insert.
2. Place slide, face up, on black backing.
3. Insert gasket into single chamber piece
4. Place on top of slide, making sure the chamber fits in the keyway of the black backing.
5. Secure assembly with two metal clips on the sides
6. Place two devices in the cytofuge 2
7. Aliquot 0.2 ml of diluted cell suspension into the chamber
8. Centrifuge slide at a setting of 6 (20g) for 6 minutes
Fixing, staining, and dehydrating slides: Standard procedure
1. Immediately after the cytofuge stops, pipet the fluid from the chamber
2. Disassemble the device, shake the excess fluid from the slide (do not allow to dry)
3. Place slides in 95% ethanol for 10 min
4. Transfer to 75% ethanol for 30 sec
5. Transfer to PBS for 30 sec.
Staining and Dehydration
1. Place cell in weighing boat and layer with 0.2 ml of PBS/10%BSA for 1 min.
2. Remove fluid and apply 100uL of primary antibody in PBS/1%BSA to slide for 10 min.
3. Wash with PBS for 2 min
4. Apply 100mL of second antibody in PBS/1% BSA and incubate for 15 min.
(in dark)
(Alternatively, replace steps 2-4 with 100mL ToPro-3 for 1-10 minutes.)
5. Wash with PBS for 2 min
6. Transfer to DI water for 30 sec
7. Transfer to 70% ethanol for 30 sec
8. Transfer to 95% ethanol for 30 sec
9. Transfer to 100% ethanol for 30 sec (100% ethanol must be completely free of water, take
aliquot directly from bottle and replace with fresh ethanol every four samples)
10. Transfer to xylene for 5 min
11. Air dry for 20 minutes