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发表于 2012-12-11 21:25:44
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Th1/Th17的Kit说明书确实有点长，今天暂时先不翻译，明后天帮你翻译一下，其实这些单词都很简单的，如果你肯用心对照词典，应该读下来也没有问题的。
A. Stimulation of the Cells
Various in vitro methods have been reported for polarization or stimulation of T helper cells subsets of which PMA (Phorbol ester) plus Ionomycin (Calcium Ionophore) has been particularly useful for quickly inducing and characterizing polyclonal cytokine-producing cells. For this kit we recommend the stimulation of normal PBMCs at a concentration of 1-10 million cells per ml in media for 5 hours with PMA/Ionomycin (at 50ng/ml and 1μg/ml respectively) in the presence of BD GolgiStop™ Protein Transport Inhibitor (provided in the kit or Cat #554724). Add 4 μl of BD GolgiStop™ for every 6 ml of cell culture and mix thoroughly. It is recommended that BD GolgiStop™ not be kept in cell culture for longer than 12 hours.
B. Staining of the Cells
1. Harvest of the Cells
Collect cells from in vitro stimulatory cultures treated with a protein transport inhibitor. Spin down cells at 250 x g for 10 minutes at room temperature (RT) and wash two times with stain buffer (FBS) (Cat# 554656). Count cells and transfer approximately 1 million cells to each flow test tube (Cat# 352008) for immunofluorescent staining. Cells should be protected from light throughout the staining procedure and storage.
2. Fixing the Cells
a. Spin down cells at 250 x g for 10 minutes at RT and thoroughly suspend cells with 1ml of cold BD Cytofix™ buffer (provided in the kit or Cat# 554655) and incubate for 10-20 minutes at RT.
Note: Cell aggregation can be avoided by vortexing prior to the addition of the fixative.
b. Spin down cells at 250 x g for 10 minutes at RT.
Note: After fixation, the cell pellet after centrifugation is loose and care should be taken when aspirating the wash buffer from the tubes. Do not aspirate ALL of the buffer but leave 50-150 μl of solution in the tubes to avoid cell loss for all subsequent wash steps below.
c. Wash cells twice at RT in stain buffer (FBS) and spin down the cells at 250 x g for 10 minutes at RT.
Note: Cells can be stored in stain buffer at 4°C for up to 72 hours or in 90% FCS/10% DMSO at -80°C for up to six months (for samples that need to be stored longer than six months, we recommend performing stability studies) .
3. Permeabilizing the Fixed Cells
a. For cells kept at 4°C, spin down cells at 250 x g for 10 minutes at RT and remove stain buffer.
b. For cells stored at -80°C thaw and wash twice with stain buffer (FBS) to remove DMSO.
c. Dilute 10x BD Perm/Wash™ buffer (provided in the kit or Cat# 554723) in distilled water to make a 1x solution prior to use.
d. Suspend cells in 1ml of 1x BD Perm/Wash™ buffer and incubate at RT for 15 minutes.
e. Spin down cells at 250 x g for 10 minutes at RT and remove supernatant.
4. Staining with the Cocktail
a. Thoroughly suspend fixed/permeabilized cells in each tube in 50 μl of BD Perm/Wash™ buffer and add 20 ul/tube of cocktail or appropriate negative control. Incubate at RT for 30 minutes in the dark. Cells should be protected from light throughout the staining procedure and storage.
b. Optional: Staining of Additional Cell Surface Antigens
Note: If instrument allows, optional multicolor staining of different cell surface antigens can be done at this time. Example: PE-Cy™7 CD3 (Clone SK7, Cat# 557851), V450 CD8 (CloneRPA-T8, Cat# 560347) and V450 CD45RA (Clone HI100, Cat# 560362) etc. For cell surface staining after fixation/permeabilization, suitable antibody clones that recognize denatured epitopes need to be identified.
Note: For antibodies that do not recognize fixed/denatured cell surface markers, it is recommended that staining be done on live cells PRIOR to fixation/permeabilization.
c. Wash cells twice with 1ml of 1× BD Perm/Wash™ buffer at RT and suspend in Stain Buffer (FBS) prior to flow-cytometric analysis.
C. Flow Cytometric Analysis
Set PMT voltage and compensation using unstained cells and appropriate cell surface markers or use BD™ Compensation beads (Cat #552843) as per the recommended protocol.
Note: It has been reported that CD4 expression on T cells is decreased after cell activation.
Note: Acquire at least 20,000 to 30,000 CD4 positive lymphocytes. Depending on the donor, frequencies of cytokine producing cells derived from activation of human PBMCs can vary widely for a particular cytokine. In order to make statistically significant frequency measurements, sufficiently large sample sizes should be acquired during flow cytometric analysis. Bivariate dot plots or probability contour plots can be generated upon data reanalysis to display the frequencies of and patterns by which individual cells co-express certain levels of cell surface antigen and intracellular cytokine proteins.
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发表于 2012-12-10 20:56:40
