I am preparing experiments to determine the superoxide content of cells (CHO and red blood cells) using hydroethidine and HPLC-MS (as described by Zielonka et al, Free radical in Biology and Medicine, 2009). However, as I am not culturing the cells, I would like to know how much cells are necessary for those analysis.
I used 6cm and 6well plates dish and methanol extraction step. See protocol below. There is about 3mil cell on 6cm and 1.2mil mil of cells in 6well plate.
Number below are for 6cm dish protocol for 6well (1mil cells) volume was reduced 3x times.
1) Put Krebs Hepes buffer in 6 cm plate
2) Add L-NAME (100uM-final) ANG II (100nM) and incubate for 30 min at 37 C
Add pg-SOD for 10 min. before you add DHE, - incubation step
3) Add DHE (10mM stock) 50uM final; 5ul to 1ml, 4ul to 800ul
4) Incubate for 1h, wash cells twice in KHB before homogenization
5) Cells are freezed homogenized in liquid nitrogen and removed to Eppendorf tubes with 100ul of 100% methanol.
6) Take 10ul aliquot of this suspension for later protein assay
7) Filter the extraction with 0.22um filter
8) Start HPLC (excitation 480nm emission 580nm; in gradient of 0.1% TFA and 60% acetonitrile in methanol) load 20ul of sample
Program:
Flow 0.45ml/min
1) 0.01 min 43% of 60% acetonitrile in methanol
2) 25 min 50.5%
3) 25.01 min 100%
4) 30 min 100%
5) 30.01 min 0%
6) 40 min 0%
7) 40.01 min 43%
8) 50 min 43%
END
Steps 3-6: wash out of column. It is necessary when used tissue or cell sample.
Retention time:
Oxyethidium 19.4 min –“superoxide” peak
Ethidium 20.6 min- reduction
I used 6cm and 6well plates dish and methanol extraction step. See protocol below. There is about 3mil cell on 6cm and 1.2mil mil of cells in 6well plate.
Number below are for 6cm dish protocol for 6well (1mil cells) volume was reduced 3x times.
1) Put Krebs Hepes buffer in 6 cm plate
2) Add L-NAME (100uM-final) ANG II (100nM) and incubate for 30 min at 37 C
Add pg-SOD for 10 min. before you add DHE, - incubation step
3) Add DHE (10mM stock) 50uM final; 5ul to 1ml, 4ul to 800ul
4) Incubate for 1h, wash cells twice in KHB before homogenization
5) Cells are freezed homogenized in liquid nitrogen and removed to Eppendorf tubes with 100ul of 100% methanol.
6) Take 10ul aliquot of this suspension for later protein assay
7) Filter the extraction with 0.22um filter
8) Start HPLC (excitation 480nm emission 580nm; in gradient of 0.1% TFA and 60% acetonitrile in methanol) load 20ul of sample
Program:
Flow 0.45ml/min
1) 0.01 min 43% of 60% acetonitrile in methanol
2) 25 min 50.5%
3) 25.01 min 100%
4) 30 min 100%
5) 30.01 min 0%
6) 40 min 0%
7) 40.01 min 43%
8) 50 min 43%
END
Steps 3-6: wash out of column. It is necessary when used tissue or cell sample.
Retention time:
Oxyethidium 19.4 min –“superoxide” peak
Ethidium 20.6 min- reduction
Dr. Zielonka did it for me and cells from one well of a confluent 6-well plate is enough. But I will suggest you go for 60 mm-plate if you want to duplicate it.
Step5 I want to kill cells quickly and make homogenate during scraping in methanol. DHE according to product specification should be kept in dark. So I try to minimaze light exposure during treatment and work. More proteins -higher signal, when cells density bettween plates is similar usually also protein content is similar and then variations are reasonoble. It is good to include positive control like LPS treated cells etc.
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