Equipment
As shown in Fig.1, the Mixer (XH-C, Yuexin Corporation, Changzhou, China) can produce the vibration in the frequency range of 0-50 Hz and with an amplitude of 3mm, and the duration can be set freely. The container filled with sample is fixed on the top of the Mixer. The parameters of processed RBCs are measured by flow cytometer (NovoCyte 2060R, ACEA Biosciences Inc., Hangzhou, China) which is shown in Fig. 2, and the NovoExpress software is applied for the data acquisition, analysis, statistics, and reporting. In flow cytometer, when the RBCs pass through the laser beams, the bigger cell has larger value of FSC which is detected by forward detector; the more irregular the cell, and/or the more protrusions on the cell surface, and/or the more organelles or granular substances in the cell, all can lead to the larger SSC value which is detected by side detectors; the antibody to the surface immune factor is conjugated to a specific fluorescein that is excited to fluoresce when it passes through the fluorescence channel of the flow cytometer.
Preparation of sample of RBCs
Anticoagulated whole blood sample which was acquired from healthy youth donors from blood bank was washed for two times by diluting with phosphate buffered saline (PBS), centrifuging for 10 min at 1500 rpm in the cryogenic high-speed centrifuge (TGL-16D, Zhongjie Corporation, Changzhou, China, shown in the Fig.3), and aspirating the supernatant. Add the PBS to the washed RBCs to make a sample of resuspended RBC solution.
Experimental procedure
The processing condition of control group (Group C) and experimental groups is shown in Table 1. The resuspended RBC solution was sent to the well plate (0.5 ml/well for 10 wells in 48 well plate; 0.25 ml/well for 10 wells in 96 well plate, as shown in Fig. 4.). The well plate with sample was then fixed to the Mixer and exposed to the vibration according to the processing condition of experimental groups which was shown in Tab.1. 0.2 ml of the processed sample was dilute with PBS, and then the photomicrograph of RBCs was taken after standing for 20 minutes. 0.1 ml of the processed sample and 1 ml PBS was transferred into a flow tube by the transferpette, and then load to the flow cytometer for shape analysis and structural complexity analysis. 1×106-3×106 RBCs were aspirated from the sample and mixed slightly with 0.1 ml the PBS, 5 μl the Mouse Anti-human IgA/FITC (BioLegend 333512), and 5 μl Mouse Anti-human IgG/PE (BioLegend 409303) (which were shown in Fig. 5), and then incubated at room temperature for 20 min in the dark; put into 1ml PBS, centrifuged at 1500 rpm for 5 min, discarded the supernatant, repeat the operation once; load to the flow cytometer for the expression of surface immune factor after mixed with 0.5 ml PBS. The experiment was repeated three times for each group and each sample was tested three times.
Table 1 The processing condition of control group (Group C) and eight experimental groups
Group No.
|
Well plate
|
Rotational speed
(rpm)
|
Duration
(min)
|
C
|
-
|
-
|
-
|
1
|
96
|
1500
|
5
|
2
|
96
|
750
|
5
|
3
|
48
|
1500
|
5
|
4
|
48
|
750
|
5
|
5
|
96
|
1500
|
10
|
6
|
96
|
750
|
10
|
7
|
48
|
1500
|
10
|
8
|
48
|
750
|
10
|