X-Gal infectivity assay
Infection
10/09/2018
o
Plate TZM-bl cells @ 2x104 cells/well in a 96 well plate (flat-bottom plate would be better).
Need 6 wells per virus, in triplicate i.e. 18 wells per virus.
11/09/2018
o
Make 10-fold dilutions of virus (50ul virus + 450ul DMEM) so the 6 wells will encompass N –
1/100,000
o
Remove media from cells and replace with 100ul of dilutions.
o
Return plate to incubator for 48rs
13/09/2018
X-gal staining
o
Remove media from cells and wash with 100ul PBS
o
Fix cells with 100µl 2% PFA* and incubate @ RT for 10-15mins
o
Remove fix and wash cells with 200ul PBS
o
Add 100ul X-gal solution† to each well
o
Incubate @ 37˚C for 2hrs (HIV-based viruses) or overnight (MLV-based viruses), then store at
4˚C.
17/09/2018
o
Wash with PBS before quantification to remove crystals
JR-FL BlaM-Vpr  10-5 dilution 8x blue cells (mean of 3wells) = 8x106 pfu/mL
JR-FL Gag-eGFP Labelled DID  10-3 dilution 12x (mean of 2wells) = 1,2x105 pfu/mL
* 2% PFA – 40 wells
†

3.5ml PBS

500µl 16% Stock PFA solution
X-gal solution – 40 wells

40µl 500mM K3[Fe(CN)6]

80µl 250mM K4[Fe(CN)6] Both are at
4˚C

8µl 1M MgCL2 (aliquots on the bench)

80µl 50mg/ml X-gal (aliquots in the
freezer -20˚C)

3.792ml PBS
If it is for imaging it should be filtered
(0.45µm)