A fixation method to maintain cell stress balance

Abstract

The invention discloses a fixation method for keeping the stress balance of cells. The method comprises the following steps: taking separation cells to be fixed, and carrying out centrifugal rinsing by using PBS; adding the PBS to a supernatant removed cell mass, mixing until uniformity, and coating the bottom of a culture dish with a cell suspension; placing the culture dish in 0DEG C environment, and irradiating by using a 254nm UV lamp at an intensity of 1.84W / cm<2>, wherein the total dose is 165.6J / cm<2>; flushing the processed cells by the PBS, and collecting the flushed cells in a centrifuge tube; and carrying out 260g centrifuging on the collected cells for 3-8min, removing the obtained supernatant, adding 4% of paraformaldehyde, fixing for 1-6s, and stopping fixation. The method guarantees the cell fixation effect, keeps the stress balance of the cells, and makes the later test result be accurate and obvious. 1-6S fixation guarantees denaturation hinge of proteins on the surface of a cell membrane and allows proteins in the cells to be in an original state; and 10s or above fixation using 4% of paraformaldehyde makes all the proteins in the cells be in a hinged state.

Description

technical field

[0001] The invention relates to a fixation method for maintaining cell stress balance. Background technique

[0002] Cells are the basic unit of life structure and life activities. The study of cell structure and intracellular chemical components and biochemical effects is an important way to reveal the physiological and pathological processes of living organisms. However, different types of living cells will undergo autolytic intracellular structural breakdown and chemical component degradation after being isolated from their living environment for a few minutes to several hours. Furthermore, the living cell membrane is a fluid, protein-embedded lipid bilayer structure, and the organelles inside the cell, including the cytoskeleton, which plays an important role in the stress structure and shape maintenance of the cell, are also in the coagulation-like state. colloidal semi-mobile phase in the cytoplasm. The behavior and structural characteristics of the ...

Examples

Embodiment 1

[0021] Detection of the effect of trypsin digestion on the binding state of cultured normal human embryonic lung fibroblasts (HFLF) SSBs (detection of 3 types: RPA32, RPA70 and POT1):

[0022] (1) Use 0.25% trypsin digestion to obtain normal human embryonic lung fibroblasts in the exponential growth phase at a cell concentration of 2×10 7 The HFLF.

[0023] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0024] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0025] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0026] (5) The collected cells were centr...

Embodiment 2

[0028] (1) Obtain mouse embryonic stem cells in the exponential growth phase with 0.25% trypsin digestion method, and the cell concentration is 2×10 7 .

[0029] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0030] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0031] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0032] (5) The collected cells were centrifuged at 260 g for 3 minutes, and after discarding the supernatant, 200 μl of 4% paraformaldehyde was added for pre-fixation for 1 second, and 46 ml of PBS was added to stop the fixation.

Embodiment 3

[0034] (1) Obtain tumor cells in the exponential growth phase with 0.25% trypsin digestion method, and the cell concentration is 2×10 7 .

[0035] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0036] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0037] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0038] (5) The collected cells were centrifuged at 260 g for 8 minutes, and after discarding the supernatant, 200 μl of 4% paraformaldehyde was added for pre-fixation for 6 seconds, and 46 ml of PBS was added to stop the fixation.

[0039] Cell in situ e...

technical field

[0001] The invention relates to a fixation method for maintaining cell stress balance. Background technique

[0002] Cells are the basic unit of life structure and life activities. The study of cell structure and intracellular chemical components and biochemical effects is an important way to reveal the physiological and pathological processes of living organisms. However, different types of living cells will undergo autolytic intracellular structural breakdown and chemical component degradation after being isolated from their living environment for a few minutes to several hours. Furthermore, the living cell membrane is a fluid, protein-embedded lipid bilayer structure, and the organelles inside the cell, including the cytoskeleton, which plays an important role in the stress structure and shape maintenance of the cell, are also in the coagulation-like state. colloidal semi-mobile phase in the cytoplasm. The behavior and structural characteristics of the ...

Examples

Embodiment 1

[0021] Detection of the effect of trypsin digestion on the binding state of cultured normal human embryonic lung fibroblasts (HFLF) SSBs (detection of 3 types: RPA32, RPA70 and POT1):

[0022] (1) Use 0.25% trypsin digestion to obtain normal human embryonic lung fibroblasts in the exponential growth phase at a cell concentration of 2×10 7 The HFLF.

[0023] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0024] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0025] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0026] (5) The collected cells were centr...

Embodiment 2

[0028] (1) Obtain mouse embryonic stem cells in the exponential growth phase with 0.25% trypsin digestion method, and the cell concentration is 2×10 7 .

[0029] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0030] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0031] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0032] (5) The collected cells were centrifuged at 260 g for 3 minutes, and after discarding the supernatant, 200 μl of 4% paraformaldehyde was added for pre-fixation for 1 second, and 46 ml of PBS was added to stop the fixation.

Embodiment 3

[0034] (1) Obtain tumor cells in the exponential growth phase with 0.25% trypsin digestion method, and the cell concentration is 2×10 7 .

[0035] (2) The cells were rinsed once by centrifugation with phosphate buffered saline (PBS). Add 200 μl of PBS to the discarded supernatant cell mass, drop it on the bottom of a 3.5 cm culture dish, and suspend the cells at the bottom of the culture dish with a pipette tip. The solution was evenly spread over the bottom of the entire culture dish.

[0036] (3) Place the petri dish on ice, use 254nm UV lamp at 1.84W / cm 2 Intensity irradiation, total dose 165.6J / cm 2 .

[0037] (4) Wash the irradiated cells in the culture dish with 20ml PBS and collect them in a 50ml centrifuge tube.

[0038] (5) The collected cells were centrifuged at 260 g for 8 minutes, and after discarding the supernatant, 200 μl of 4% paraformaldehyde was added for pre-fixation for 6 seconds, and 46 ml of PBS was added to stop the fixation.

[0039] Cell in situ e...

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