Ficoll density gradient centrifugation separates individual nuclear cells of outer blood.

to determine cellular immune function, active cells, such as lymphocytes, macrophages, granulocytes, etc., are first obtained from human or animal exoglomes or
tissues
. Active cells should be obtained according to different purposes, using different methods, considering
(1) cell purity,
(2) cell yield,
(3) cell vitality,
(4) ease of use and room conditions.
currently commonly used Ficoll density gradient centrifugation method to directly isolate and purify the outer blood of a single nuclear cell.
(i) Principle:
is commonly used to isolate a single nuclear cell (PBMC) of human exomens with a strated fluid specificity of 1.077±0.001 of ficosaccharide (Ficoll)-Urografin (F/H) strated fluid. Ficoll is a polymer of sucrose, neutral, W high water quality, with an average molecular weight of 400,000, when the density of 1.2g/ml still does not exceed the normal physiological osmosis pressure, nor through the biofilm. Red blood cells, granulocytes than significant, centrifugal sink to the bottom of the tube, lymphocytes and monocytes of the proportion of less than or equal to the proportion of strated fluid, centrifugal floating on the liquid surface of layered liquid, can also have a small number of cells suspended in layered liquid. Cells that absorb strated liquid surfaces can be isolated from outer blood to a single nucleocyte.
(ii) Method:
. 1. Add an appropriate amount of lymphocyte separation fluid to the short medium tube.
2. Take heparin anticoagulant vein blood and the equivalent Hank's liquid or RPMI1640 fully mixed, with a dropper along the pipe wall slowly superimposed on the layered liquid surface, pay attention to maintain a clear interface. Horizontal centrifuge 2000rpm × 20 minutes.
3. The centrifugal tube is divided into three layers, the upper layer is plasma and Hank's liquid, the lower layer is mainly red blood cells and granulocytes. The middle layer is the lymphocyte separation fluid, and at the upper and middle interfaces there is a narrow band of white clouds dominated by individual nuclear cells (see figure below), with individual nuclear cells including lymphocytes and monocytes. Plateplates are also included.
4. Use capillaries to insert into the clouds and absorb individual nuclear cells. Place in another short medium tube, add more than 5 times the volume of Hank's liquid or RPMI1640,1500rpm× 10 minutes, wash the cells twice.
5. After the last centrifuge, discard the qing and add RPMI1640 containing 10% calf
serum
, re-hanging cells. Take a drop of cell suspension mixed with a drop of 0.2% table blue dye and count the total number of cells in four large squares on the blood cell counting board.
Single nuclear cell concentration (cell count /1 ml cell suspension) -
total number of cells in a large square
-- -- × 104×2 (dilution multiple)
4
6. Cell vitality detection: dead cells can be dyed blue, live cells do not color. Count 200 lymphocytes. Calculate the percentage of living cells.
Number of living cells
Percentage of living cells -- ×100%
Total number of cells
the PBMC is isolated by this method, purity is above 90%, harvest rate can reach 80 to 90%, and percentage of living cells is above 95%.
.