Flow Cytometry Analysis using Peripheral Blood Cells:
1) Collect approximately 75 microliters of blood into 1mL PBS containing 5microM EDTA and mix immediately to prevent clotting. keep the tubes on ice.
(10 microliters of 0.5M EDTA stock).
2) Remove Red blood cells from samples. [RBCs are lysed using either Gey's solution or a buffered ammonium chloride solution. (Or, BD sells a product called FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells)]. Use proper solution according to the instructions given by the manufacturer.
3) Wash the cells at least 2-3 times with FACS buffer. (FACS buffer contains PBS solution supplemented with either 1% BSA or 5% FBS and 0.05% NaN3).
4) Suspend the pellet from the final wash in 50microliters FACS buffer per each analysis on a single sample. (Up to three separate staining reactions can be set up from a single sample).
5) Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently. (You need to make sure to determine the proper concentration for each antibody used using Antibody Titration).
6) Keep the tubes on ice for 30 minutes for incubation.
7) Using FACS buffer, wash the cells 2 to 3 times and suspend in 200-300 microliters FACS buffer for analysis purpose.
8) Make sure to have samples with minimum concentration at least million per ml.
Example of the peripheral blood analysis using flow cytometry is shown below.
Source: [Paper: Pregnancy and Preeclampsia Affect Monocyte Subsets in Humans and Rats]
1) Collect approximately 75 microliters of blood into 1mL PBS containing 5microM EDTA and mix immediately to prevent clotting. keep the tubes on ice.
(10 microliters of 0.5M EDTA stock).
2) Remove Red blood cells from samples. [RBCs are lysed using either Gey's solution or a buffered ammonium chloride solution. (Or, BD sells a product called FACS lysis buffer" that is used after the staining protocol to lyse RBCs and fix the cells)]. Use proper solution according to the instructions given by the manufacturer.
3) Wash the cells at least 2-3 times with FACS buffer. (FACS buffer contains PBS solution supplemented with either 1% BSA or 5% FBS and 0.05% NaN3).
4) Suspend the pellet from the final wash in 50microliters FACS buffer per each analysis on a single sample. (Up to three separate staining reactions can be set up from a single sample).
5) Add 50 microliters of cell suspension to 10 microliters of antibody solution and mix gently. (You need to make sure to determine the proper concentration for each antibody used using Antibody Titration).
6) Keep the tubes on ice for 30 minutes for incubation.
7) Using FACS buffer, wash the cells 2 to 3 times and suspend in 200-300 microliters FACS buffer for analysis purpose.
8) Make sure to have samples with minimum concentration at least million per ml.
Example of the peripheral blood analysis using flow cytometry is shown below.
Source: [Paper: Pregnancy and Preeclampsia Affect Monocyte Subsets in Humans and Rats]
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| Research Gate: Pregnancy and Preeclampsia Affect Monocyte Subsets in Humans and Rats |
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