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After fixing cells in ethanol it was determined that HIV-1 was inactivated by
measuring p24 in co-cultures for 36 days. This was controlled by both
untreated (unfixed) and PFA fixed samples. We sorted cells for RT-PCR and our
sorting strategy worked very well. Below is our fixation procedure but first a
few thoughts to keep in mind "pre-sort".
a. Run ethanol (70% in DEPC) through system prior to sort for at least 5 mins.
b. HOH in sheath should be DEPC treated.
c. Sort tip should be wiped down with ethanol and with vacuum on should be
allowed to suck though tip.
Procedure for Ethanol fixation of PBL's:
1. F/H separated blood was washed 2x in PBS-DEPC.
2. To 15 x 106 cells re suspended into 500 l of PBS-DEPC. To this was added
5ml of Et-OH (70% ethanol in DEPC-HOH *) drop by drop while vortexing.
3. Incubate mixture 30min. at 4oC
4. Wash 2x in PBS-DEPC and re suspend in PBS-DEPC.
* DEPC = Diethyl pyrocarbonate : 1ml per 1 liter of HOH, 100 oC for 15min. to
evaporate all DEPC as CO2. This treatment effectively removes all RNase from
any contamination source. Note diluted PBS was also prepared with DEPC water.
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