John M. S. Bartlett.pdf - Bio-Nica.info

448 Gilchrist and Befus
2. Cells are pelleted by centrifugation in a tabletop centrifuge at 800g for 5 min at 4°C.
3. Cells should be washed once by resuspending in 10 mL of PBS and centrifuged again
at 800g for 5 min at 4°C.
4. The supernatant is discarded, and the pelleted cells are then fixed in 10 mL of 10% neutral
buffered formalin for 16 h at room temperature (see Note 2).
5. After fixation the cells are twice washed in DEPC water, recovering the cells each time by
centrifugation at 800g for 5 min at 4°C.
6. The cells are then resuspended in DEPC water.
7. Cells are then dropped (75 µL) onto slides in three discrete areas and allowed to air dry
at room temperature. Cell concentration should be between 5000 to 10,000 cells per
droplet/spot. (Fig. 2) (see Note 3).
3.2. Protease Digestion
1. Dissolve 20 mg of pepsin in 9.5 mL of DEPC-treated water and then add 0.5 mL of 0.2 N
HCl. The final concentration of pepsin should be 5000 U/mL.
2. Add 200 µL of this mix to each spot on the slide (see Note 4). The digestion time has
to be standardized for each cell type (see Note 5). For mast cell lines we use a digestion
time of 45 min at 37°C.
3. After the digestion is completed, the pepsin is inactivated with a 1-min wash in DEPC
water in a Coplin jar.
4. Then, wash the slides in 100% ethanol for 1 min.
5. Allow to air dry at room temperature (see Note 6).
3.3. Pretreatments (Optional)
In our studies of mast cells, the presence of granule-associated heparin in most
preparations is a major obstacle (9). Heparin is a potent inhibitor of RT and Taq
polymerase enzymes, and therefore a heparinase digestion step must be included to
remove heparin before proceeding.
1. 50 µL of heparinase digest buffer containing 333 U/mL of heparinase I is added to the
slide and incubated for 2 h at room temperature.
2. Wash the slides in DEPC water for 1 min.
3. Then wash the slides in 100% ethanol for 1 min.
4. Allow the slides to air dry at room temperature.
3.4. DNase Treatment
This step is used to remove chromosomal DNA. It is added to the negative control
and test cell spots only. Complete removal of genomic DNA is essential for correct
interpretation of the test cells.
1. The negative control and the test cell spots are treated with 50 µL of DNase solution per
spot, and incubated overnight at 37°C.
2. After the overnight digestion, wash the slides in DEPC water for 1 min.
3. Then wash the slides in 100% ethanol for 1 min.
4. Allow the slides to air dry at room temperature.
3.5. Reverse Transcription
1. Add 50 µL of the master mix to the test spot ONLY (see Note 7).
2. Incubate 1 h at 37°C in a moist chamber. Transcripts of low abundance may require longer
incubation of the RT step (see Note 8).