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Flow Cytommetry The flow cytometer uses a laser beam and light detector to count single intact cells in suspension. The simplest form of the instrument counts each cell as it passes the laser beam and record the level of fluorescence the cell emits; an attached computer generates plots of the number of cells as the ordinate and their fluorescence intensity as the abscissa. Cells labeled with fluorochrome conjugated antibody can be analyzed and sorted on the basis of intensity of fluorescent antibody staining in a specialized flow cytometer called a fluorescence activated cell sorter (FACS). One problem with amplification of macromolecules by immunocytochemistry and the like is unbiasing when trying to quantify how much of an object is present. Seterology introduces a number of rules to avoid this bias. One major rule is systematic random sampling. Understanding the Results
upper squares tell you presence of antibodies on Y axis right side 2 squares are for X axis
Flow cytometric immunophenotyping is based on the identification of single or multiple surface membrane antigens or intracellular antigens of cells in suspension simultaneously with the light scatter characteristics of these cells. The antigens are detected with fluoresceinated monoclonal antibodies. Non-nucleated cells are depleted before analysis. Basic procedure: of Cytokine flow cytometry (CFC): involves short-term in vitro activation (frequently 6 h) with protein or peptide antigens or mitogens, in the presence of a secretion inhibitor such as brefeldin A to enhnace the accumulation of cytokines or other intracellular markers. (e.g., To prevent cytokine secretion, befeldin A (10 ug/ml/106 cells) (Sigma-Aldrich) can be added to cell cultures during the last 24 h of stimulation (day 7)). For peptides or mitogens which do not require processing by host antigen-presenting cells, the secretion inhibitor can be present during the entire incubation period with no reduction in response. For protein atnigens, an anitial period (1-2 h) of incubation is performed with antigen alone to allow for optimal antigen processing and presentation which could otherwise be compromised by the presence of brefeldin A or monensin. The entire incubation peirod is deliverately short to main cell viability and to accumrately capture the frequency of responsive cells in whole blood or PBMC samples. Sample Protocol for FACS: Station/Cell Preparation (1) prepare station (white towel; Prepare 2% FCS in PBS (ex. .4 ml FCS for 20 ml, 0.5 ml for 25 ml, .6 ml for 30 ml, 1 ml for 50 ml, and 2 ml for 100ml). Obtain 200, 20 and 2 ul pipettes, steril 10 and 200 ul tips and alcohol swabs (used to clean the pipettes). Obtain antibodies FITC (green cap), 2nd PE (red cap) 3rd PERCP 4th APC (blue cap)) and place in small beaker and put this on ice. (2) Your cells will have been treated (e.g., 24 hours) in 15 ml tubes. Generally, you will need 1x10e6 cells per tube. But FACS can be done with less (e.g., could use 05. x 10e6 cells. So if know you are going to have 5 FACS tubes would want 2.5 ml of cells at concentration of 1x10e6). Spin cells down. (3) Get clear plastic tubes with plastic caps (FACS tubes, 3rd drawer). We will have separate tubes (1 for control and 1 for each of the antibodies used) for each of our experimental groups. For example, if treatment groups are --DCs only, DC+LPS, DC+LPS+EGCG and we want to look at 4 markers, we would have 5 separate tubes for each of these 3 groups. Label these tubes (group name (e.g., DC only, DC+LPS, DC+LPS+EGCG); If control lable "control" Control will have no antibodies. Also label the name of the antibody used (eg. FITC-CD11c/ PE-CD11b/APC-F480) (e.g., CD11cFITC/CD40PE, CD11cFITC/CD80PE, CD11cFITC/CD86PE, CD11cFITC/FLII-IA-PE, Control). Flame the writing so it stays on. Place tubes on ice. Up to 4 different types of Ab can be used in any one same tube. (4) Remove supernatent from 15 ml tubes. Resuspend cells in your 2% FBS. Use 100 ul per number (e.g., 1 milliong, 0.5 million) of cells. (e.g., if you want 0.5 x 10e6 cells in 100 ul in each FACS tube your would need to use 500 ul for 5 tubes) (e.g., You have 5 million cells per tube. Each million has to go to 100 ul. So would use 500 ul) Block nonspecific Binding (1) Add 4 ul per 106 cells. (e.g., if have 2-2.5 million cells in orange tubes on ice add 8 ul) (e.g., if have 5x10e6 cells in the orange tube, add 20 ul) (FC is blue cap (CD16/CD32). Let this sit for 5 minutes. Antibodies (1) While cells are incubating with FC antibody on ice do the following: Line up all your FACS tubes per group but so the antibodies are matched in rows. (e.g., control (no antibodies, not even CD11c, CD11c/CD86, CD11c/CD40, CD11c/CD80, CD11c/CD80). 1st add any common antibodies to each of the tubes (e.g., CD11c). Use 3-4 ul of the marker. (3) take 100 ul of cells from 15 ml tube which has been resuspended in 2% BGS (1st place 100 ul in control and then place into other tubes according to the respective groups. (go group by group). (4) Incube 30 min (cover ice bucket w/ lid because this needs to be done in the dark!) Wash Cells (1) Add 2 ml of the 2% PBS solution to each tube. (2) Change holders in centrifuge to yellow and spin down. Align tubes so they are directly diagonal. Peraformaldyhde treatment (1) prepare peraformadyhyde (keep flask covered with aluminum foil). 1st prepare 4%. paraformaldyhde (if powder in the fridge). --take a flask and place magnet in it (e.g., prepare 25 ml; use 125 ml flask) --add 25 ml of PBS --warm it up on hot plate (just turn to like 100 and let warm. should be about 63C) --add 1g of peraformadehyde poweder, stir with small magent until clear --cool it down on ice. To prepare 1%--use 1 ml of the 4% and 3 ml of PBS. Label 50 ml tubes this can be kept for 2 weeks. (3:1; 6:2; 9:3; 12:4; 15:5; 18:6; 21:7) (2) gently remove supernatent from tubes (use paper to get it out at end). Vortex. (3) Add 1 ml for each 10e6 cells of your 1% peraformadelhyde solution. vortex again. (4) wrapp tubes in aluminum foil and place in fridge.
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