I am working on comparative study of cytokines secreted during cancer growth in cancer patients. I will extract the samples and would perform HPLC and OD for estimation of cytokines.
Dear Sonali,
blood samples:
I suggest to collected 4 mL peripheral blood in anticoagulant EDTA tube (BD Vacutainer®) and 9 mL in sodium heparin tube (BD Vacutainer ®) for cell culture, processed less than 3H.
Peripheral blood mononuclear cells (PBMC) isolation and cellular culture
PBMC shoul be isolated from heparinized blood by 3mL Isolymph® 1077 (Sigma, St. Louis, MO, USA) density gradient centrifugation (250g for 30 minutes at 21 °C). PBMCs were washed (PBS 1x pH 7,4) and centrifuged (250g for 5 minutes at 21 °C) tree times. 1x106 cells per well in 200 µL with RPMI supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µL streptomycin and 2nM L-glutamine (Sigma, St.Louis, MO, USA). Cells sample shoul be incubated with 2 µL phytohemaglutin (PHA, 10 µg/mL) (Sigma, St.Louis, MO, USA) per well in triplicates and incubated at 37 °C for 24H in a CO2 incubator (Sanyo Electric Biomedical Co., Ltd, USA). After 24H, supernatant was frozen at -80 °C for cytokines analysis.
I agree with Juliana that PBMC are probably the best idea to assess cytokines in cancer patients, though this may vary depending on the tumor location and what you are trying to see. Some epithelial and stromal cells also produce cytokines, as do fibroblasts and some other cells that may be missed from the PBMCs. The answer is going to depend a lot on what cytokines you are hoping to see and which type of cancers you are looking at.
As a second thought, since different subsets of immune cells (which will be captured in your PBMC isolation) also produce different kinds of cytokines, you may wish to consider separating out some of the different immune lineages (T-cells, B-cells, DCs for example).
Blood and then isolate PBMC from the blood.. Le me know if u need protocol for that
I am really thankful to Juliana, Amanda and Gaurav.
Further I would like to mention that I am working on cervical cancer patients and will also be comparing the cytokine levels in them during fungal infections.
Weather PBMC would be able to cover the epithelial and Mast cell associated cytokines that work in the initial immunity phase against fungal infections or not??
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