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Microrganism survival following uptake by DCs
Laboratory protocolAfter 6 hour of stimulation, DCs were collected, washed 3 times with PBS, treated with zymolyase, washed twice and cells, lysated with a hypotonic solution (KCl 0.05%), were plated on YPD. Survival of yeast cells, spores or hyphae after uptake was reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure. To assess the importance of ROS production in DC killing ability, DPI (10 microM) was added 30 minutes before stimulation and survival of microorganisms was assessed using the same method. When evaluating survival after exposure, the possible effect of DMSO and DPI on the stimuli was taken into account.
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Stimulation of DCs
DCs are stimulated over 6 hours.
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Washing of DCs
DCs are collected and washed 3 times with PBS.
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Treatment of DCs
DCs are treated with zymolyase.
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First washing of DCs
DCs are washed.
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Second washing of DCs
DCs are washed.
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Plating of DCs
Cells, lysated with a hypotonic solution (KCl 0.05%), are plated on YPD.
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Exposure and assessment of survival of yeast cells, spores or hyphae after uptake
Survival of yeast cells, spores or hyphae after uptake is reported as percentage of colony forming units after 3 days relative to the total number of cells growing in the absence of DCs exposure.
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Exposure and assessment of importance of ROS production in DC killing ability
When evaluating survival after exposure, the possible effect of DMSO and DPI on the stimuli is taken into account.
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Addition of DPI
To assess the importance of ROS production in DC killing ability, DPI (10 microM) is added 30 minutes before stimulation.
created over 15 years ago
(21 January 2010)
last modified over 13 years ago
(28 September 2011)
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