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University
of Washington School
of Medicine, Department
of Microbiology
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| Determining
Coreceptor Usage of HIV-1 on U87 Glioma Cells |
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Reagents:
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U87
T4 co-receptor cell lines (mycoplasma free):
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CD4
only
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CD4
plus CCR1
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CD4
plus CCR2b
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CD4
plus CCR3
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CD4
plus CCR5
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CD4
plus CXCR4
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cDMEM
(DMEM, 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin, L-Glutamine, 10mM HEPES)
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Sterile
TC 1x PBS.
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1x
Trypsin.
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Geneticin
(G418), 300 µg/ml
final concentration.
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Puromycin,
1 µg/ml
final concentration.
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96-well
microtiterplate with lid (flat bottom), Nuclon, Nunc.
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24-well
plate with lid (flat bottom), Costar?
Passaging:
U87 cell stocks are cultured in T25 flasks in 7ml
cDMEM with G418 for all cell lines and puromycin for all
except parental U87 T4.
For passaging, wash monolayer with 5ml warm PBS.
Add 0.5ml 1X Trypsin.
Incubate 2 minutes at room temperature.
Confirm that all cells detach.
Add 2ml cDMEM and pipette up and down to
completely resuspend all cells.
Use 250 µl
cell suspension to continue culture. Add 7ml cDMEM and
antibiotics.
Pellet cells not used for passaging and resuspend
at appropriate concentration for further use (i.e. 1 x
105/ml for co-receptor assay).
Co-receptor
assay:
Per virus per cell line: plate out 1 x 104
cells (100 µl)
in cDMEM with antibiotics in 96-well plate.
Inoculate with at least 100 TCID50 (in
no more than 100 µl).
After 24 hours wash with warm PBS.
Add 200 µl
fresh cDMEM with antibiotics.
After 3 and 7 days, take p24 sample. Store at
-20°C.
After 7 days, score cytopathic effect.
Wash monolayer with warm PBS.
Add 50 µl
1x Trypsin.
Incubate 2 minutes at room temperature.
Transfer each well to a new well in a 24-well
plate.
Add 2 ml cDMEM with antibiotics
After 10, 14, 17 and 21 days, take p24 samples.
Store at -20°C.
After 14 and 21 days, score cytopathic effect.
Assay p24 production and discard cultures.
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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