A fluorescence intensity correction method and cell concentration detection method of a mitochondrial probe
A technology of fluorescence intensity and cell concentration, applied in fluorescence/phosphorescence, measurement devices, material analysis through optical means, etc., can solve problems such as inability to express mitochondrial mass values, decrease in fluorescence intensity values, and increase in binding areas
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Embodiment 1
[0061] 1. Establish a Logit correction model
[0062] 1. A peripheral blood sample was diluted into 7 concentration points according to the white blood cell concentration, which were 3.4×10 3 , 5.1×10 3 , 7.6×10 3 , 11.4×10 3 , 17.1×10 3 , 25.6×10 3 unit / μL, select the target cell group naïve CD4+T (CD4+CD45RA+CD62L+) to analyze the mitochondrial fluorescence intensity value of this group. The original data results are shown in Table 1 below:
[0063] Table 1
[0064]
[0065] 2. Establish a Logit correction model according to the original data: log(Y)=nlog(X)+a; linear regression, the obtained results are as follows figure 1 shown. Among them, a is a constant, and the value of n obtained by this model is as follows: n(CD3) = -0.8~-0.5.
[0066] 3. The fluorescence intensity correction value y of the sample to be tested is deduced according to the Logit correction model k :
[0067]
[0068]
[0069] where y k is the corrected value of fluorescence in...
Embodiment 2
[0074] 1. Establish a Logit correction model
[0075] Three samples were stained and labeled with CD3 FITC and mitochondrial probe, which were marked as sample 1, sample 2, and sample 3; for sample 1, cells were counted using absolute counting microspheres equipped with a flow cytometer. The results are shown in Table 3.
[0076] table 3
[0077]
[0078] see Figure 4 , build a Logit correction model based on the original data: log(Y) = -0.6964log(X)+5.5849.
[0079] 2. Calculate the cell concentration through the Logit correction model
[0080] Dilute sample 2 by 2 points and record it as unknown concentration point C11 and unknown concentration point C12; dilute sample 3 by 2 points and record it as unknown concentration point C21 and unknown concentration point C22; refer to the absolute counting method of sample 1 to detect the above four points. The absolute cell count value x at each unknown concentration point.
[0081] Samples 2 and 3 were both stained and labe...
Embodiment 3
[0085] Example 3 Establishing the Logit correction model of the cell concentration value X and the fluorescence intensity value Y of the standard cell line
[0086] 1. Cell Sample Preparation
[0087] 1.1 The cell laboratory is routinely sterilized, irradiated with ultraviolet light for more than 30 minutes, and the ultra-clean bench is turned on for ventilation for 10 minutes.
[0088] 1.2 The cell culture medium (1640+10% FBS) was preheated at 37°C, and wiped and disinfected with 75% alcohol before moving into the cell ultra-clean bench.
[0089] 1.3 Take out the cell cryovial from the liquid nitrogen tank, immediately place it in a 37°C water bath and shake the cell cryotube quickly to thaw it quickly.
[0090] 1.4 Transfer the cells in the cryopreservation tube into a 15mL centrifuge tube in an ultra-clean bench, add 3mL of fresh cell culture medium, and centrifuge at 800 rpm for 5 min.
[0091] 1.5 During the centrifugation waiting time, in the cell culture flask (25cm ...
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