This plan only provides guidance, please make modifications according to your specific needs.
1. Cell collection
(1). Suspension cells: Collect approximately 105-106 cells, centrifuge at 4℃ and 1000g for 3-5 minutes, carefully aspirate the supernatant, add 1mL of pre cooled PBS to resuspend the cells, and transfer to a 1.5mL centrifuge tube. Centrifuge the cells again and carefully discard the supernatant. Approximately 50μL of PBS can remain to avoid removing the cells.
(2). Adhering cells: Suck out the cell culture medium and transfer it to a centrifuge tube of appropriate size. Gently wash the adherent cultured cells with PBS and add an appropriate amount of trypsin cell digestion solution to digest the cells. Incubate at room temperature until the cell shape gradually becomes round and loose, and gently shake until cell detachment occurs. Immediately add the collected cell culture medium to the digested cells to terminate digestion, gently blow down the cells, and collect them in the same centrifuge tube. Centrifuge at 4℃ and 1000g for 3-5 minutes, carefully discard the supernatant, add 1mL of pre cooled PBS to resuspend the cells, and transfer to a 1.5mL centrifuge tube. Centrifuge the cells again and carefully discard the supernatant. Approximately 50μL of PBS can remain to avoid removing the cells.
(3). Tissue cells: Cut the tissue into as small pieces as possible with scissors, digest with trypsin for 0.5-1 hour, and filter through a 200-400 mesh sieve to obtain a single-cell suspension. Take about 105-106 cells, centrifuge at 4℃ and 1000g for 3-5 minutes, carefully aspirate the supernatant, add 1mL of pre cooled PBS to resuspend the cells, and transfer to a 1.5mL centrifuge tube. Centrifuge the cells again and carefully discard the supernatant. Approximately 50μL of PBS can remain to avoid removing the cells.
2. Cell fixation and rinsing
Add 1mL of pre cooled 70% ethanol to the cells, gently blow and mix well, and fix at 4℃ for 2 hours or longer. After fixation, collect the cells by centrifugation at 1000g for 3-5 minutes, carefully aspirate the supernatant, add about 1mL of pre cooled PBS, and resuspend the cells. Centrifuge the cells again and carefully discard the supernatant.
Note:
(1). It is recommended to fix for 2 hours or more to ensure the staining effect, and fix for 12-24 hours for better results.
(2). When fixing cells, the following scheme can also be used: first add 300μL PBS to the cell pellet, resuspend and disperse the cells thoroughly, then add 700μL anhydrous ethanol and mix well to obtain a 70% ethanol fixative, which can avoid cell clustering or adhesion.
(3). For specific cells, if the cell precipitation is incomplete after fixation and centrifugation, it is recommended to extend the centrifugation time or increase the centrifugal force appropriately until the cells are completely precipitated.
(4). After cell fixation, the volume will become lighter and easier to suspend during centrifugation. PBS containing 2% BSA can be replaced with PBS to assist cell sedimentation.
(5). When removing the fixative and rinsing the cells, about 50μL of the supernatant can be retained each time to avoid discarding the cell sample.
3. Cell staining
(1). Refer to the table below and configure the staining solution according to the sample size:
|
|
1 sample
|
6 sample
|
12 sample
|
|
Cell Stain Buffer
|
0.5mL
|
3mL
|
6mL
|
|
PI Stain Buffer
|
10μL
|
60μL
|
120μL
|
|
RNase A
|
10μL
|
60μL
|
120μL
|
|
Total Volume
|
0.52mL
|
3.12mL
|
6.24mL
|
Note: It is recommended to use the staining solution immediately and prepare it on the same day.
(2). Add 0.5mL of PI staining solution to each tube of cell samples, gently resuspend the cells, and incubate at 37℃ in the dark for 30 minutes. After incubation, it can be stored at 4℃ or in an ice bath away from light. It is recommended to start the machine as soon as possible, preferably completing the sample flow testing within 2-5 hours.
4. Flow detection
Use flow cytometry to detect fluorescence, and perform cell DNA content analysis and light scattering analysis using appropriate analysis software. The maximum excitation/emission wavelength of PI binding to nucleic acid is 535/617nm.
Note: Before starting the machine, use a 200-400 mesh sieve to filter the cell suspension and obtain dispersed individual cells.
