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Principle of experimental method:
of 1.
10% DMSO or glycerol freezing medium preparation of 10~20% calf serum
.
Trypsin-digested logarithmic cells during the growth phase, with cells grown in suspension moved directly to a 15 ml centrifuge tube
.
3.
Centrifuge speed 1000 rpm, 5 minutes
.
4.
Remove trypsin and old medium and add an appropriate amount of prepared frozen medium
.
Gently inflate the cells with a pipette to evenly and count, adjust the final density of cells in the cryolyte to 5×106/ml to 1×107/ml
.
Divide the cells into deep cryopreservation tubes, 1~1.
5 ml
per tube.
。
6.
Indicate the cell name, freezing time and operator on the cryopreservation tube
.
。
7.
Freezing: The standard freezing procedure is that the cooling rate is -1 ~-2 °C/min
.
When the temperature is lower than -25 °C, it can be increased to -5 °C~-10 °C /min.
。 At -100 °C, it can be quickly immersed in
liquid nitrogen.
。 Cryopreservation tubes containing cells can also be placed in a refrigerator at -20 °C for 2 hours and then placed in a -70 °D refrigerator overnight
.
Remove the cryovial and move it into a
liquid nitrogen container.
。 III.
Cell Recovery1.
Remove the cryovials from the liquid nitrogen container and immerse them directly in warm water at 37 °C, shaking from time to time to allow them to melt
as soon as possible.
。
Remove the cryopreservate tube from the 37 °C water bath, open the lid, aspirate the cell suspension with a pipette, add a centrifuge tube to drop the culture medium more than 10 times, stir well
.
。
Centrifuge at 1000 rpm for 5 min
.
Discard the supernatant, add 10% calf serum medium to suspend the cells, count, adjust the cell density, inoculate the flask, and culture
in a 37 °C incubator.
5.
Change the medium the next day and continue the culture
.
。 Registration Matters:
1.
Cultured cells from the proliferation phase to the formation of dense monolayer cells can be used for cryopreservation, but logarithmic growth phase cells
are preferred.
It is best to change the medium
the day before freezing.
2.
When placing the cryogenic storage tube into a liquid nitrogen container or removing it, do a good job of protection to avoid frostbite
.
。
3.
For cryo-resuscitation, it is best to use newly formulated medium
.
The basic principle of cell cryopreservation and recovery is slow freezing and fast thawing, which has been shown to maximize cell viability
.
Glycerol or dimethyl sulfoxide are commonly used as protective agents for cellular cryopreservation
.
These two substances can improve the permeability of cell membranes to water, and slow freezing can make water in cells exude from cells, reducing the formation of ice crystals in cells, thereby reducing the damage
to cells caused by ice crystal formation.
Rapid melting should be used to ensure that extracellular crystals melt in a short period of time to avoid damage
to cells due to the slow melting of water into cells to form intracellular recrystallization.
Experimental steps:First, cell cryopreservation.
Glycerol or dimethyl sulfoxide are commonly used as protective agents for cellular cryopreservation
.
These two substances can improve the permeability of cell membranes to water, and slow freezing can make water in cells exude from cells, reducing the formation of ice crystals in cells, thereby reducing the damage
to cells caused by ice crystal formation.
Rapid melting should be used to ensure that extracellular crystals melt in a short period of time to avoid damage
to cells due to the slow melting of water into cells to form intracellular recrystallization.
of 1.
10% DMSO or glycerol freezing medium preparation of 10~20% calf serum
.
Trypsin-digested logarithmic cells during the growth phase, with cells grown in suspension moved directly to a 15 ml centrifuge tube
.
3.
Centrifuge speed 1000 rpm, 5 minutes
.
4.
Remove trypsin and old medium and add an appropriate amount of prepared frozen medium
.
Gently inflate the cells with a pipette to evenly and count, adjust the final density of cells in the cryolyte to 5×106/ml to 1×107/ml
.
Divide the cells into deep cryopreservation tubes, 1~1.
5 ml
per tube.
。
6.
Indicate the cell name, freezing time and operator on the cryopreservation tube
.
。
7.
Freezing: The standard freezing procedure is that the cooling rate is -1 ~-2 °C/min
.
When the temperature is lower than -25 °C, it can be increased to -5 °C~-10 °C /min.
。 At -100 °C, it can be quickly immersed in
liquid nitrogen.
。 Cryopreservation tubes containing cells can also be placed in a refrigerator at -20 °C for 2 hours and then placed in a -70 °D refrigerator overnight
.
Remove the cryovial and move it into a
liquid nitrogen container.
。 III.
Cell Recovery1.
Remove the cryovials from the liquid nitrogen container and immerse them directly in warm water at 37 °C, shaking from time to time to allow them to melt
as soon as possible.
。
Remove the cryopreservate tube from the 37 °C water bath, open the lid, aspirate the cell suspension with a pipette, add a centrifuge tube to drop the culture medium more than 10 times, stir well
.
。
Centrifuge at 1000 rpm for 5 min
.
Discard the supernatant, add 10% calf serum medium to suspend the cells, count, adjust the cell density, inoculate the flask, and culture
in a 37 °C incubator.
5.
Change the medium the next day and continue the culture
.
。 Registration Matters:
1.
Cultured cells from the proliferation phase to the formation of dense monolayer cells can be used for cryopreservation, but logarithmic growth phase cells
are preferred.
It is best to change the medium
the day before freezing.
2.
When placing the cryogenic storage tube into a liquid nitrogen container or removing it, do a good job of protection to avoid frostbite
.
。
3.
For cryo-resuscitation, it is best to use newly formulated medium
.
