Rat microglia extraction strategy

1. Preparation before the experiment

 

1) Animals:

The whole brain (including cortex, hippocampus and striatum) of newborn 1-3 days old SD rats.

 

2) Main reagents:

• DMEM (abs9483)

• Fetal bovine serum (abs972)

• Double antibody (abs9244)

• 0.25% trypsin-EDTA (abs47014938)

• Tissue dissociation solution (abs9482)

• 10xPBS (abs961)

• 1xPBS (abs962)

• Percoll (abs9102)

• 70um cell screen (abs7232)

• Cell culture dish (35mm) (abs7004)

• Cell culture dish (60mm) (abs7005)

 

2. Microglia extraction and sorting

 

• Enzymatic digestion

(1) Cut the brain tissue into small tissue pieces of 0.5-1mm3, transfer the tissue pieces (100-200mg) to a 2mL round-bottomed tube, add 1mL of digestive juice (cat: abs9482), and gently shake and incubate at a speed of 80rpm at 37 °C (extend the time appropriately according to the tissue erosion state)

(2) Take out the round-bottom tube from the horizontal shaker, swirl the tube vigorously for 10-20s, filter through a 70um cell sieve (cat: abs7232), and rinse the cell sieve with 10mL PBS;

(3) Collect the filtrate containing cells into a 50mL tube, centrifuge at 400g at 20 °C for 5min, and discard the supernatant.

 

• Percoll density gradient purification

(1) 30% Percoll solution

Percoll cell separation solution was mixed with PBS buffer (10 ×) at a volume ratio of 9: 1 to prepare an isotonic 100% Percoll mother liquor.

A 30% isotonic Percoll solution was prepared by mixing 100% Percoll mother liquor and 1 × PBS at a volume ratio of 3: 7.

 

(2) 37% Percoll solution

Perco11 cell separation solution was mixed with PBS buffer (10x) at a volume ratio of 9: 1 to prepare an isotonic 100% Percoll mother solution. 100% Percoll mother solution was mixed with 1x PBS at a volume ratio of 37: 63 to prepare a 37% isotonic Percoll solution.

 

(3) 70% Percoll solution

Perco11 cell separation solution was mixed with PBS buffer (10 ×) at a volume ratio of 9: 1 to prepare an isotonic 100% Percoll mother solution. 100% Percoll mother solution was mixed with 1 × PBS at a volume ratio of 7: 3 to prepare a 70% isotonic Percoll solution;

 

(4) Pipette 4 mL of 70% percoll deep into the bottom of a 15 mL centrifuge tube;

 

(5) Resuspend the cell pellet with 4 mL, 37% percoll (cat: abs9102), slowly add 4 mL of 50% percoll centrifuge tube along the tube wall, and cover the top of 70% Percol solution to form a density gradient;

▲ CAUTION: Do not destroy the interface between 37% percoll and 70% percoll and the suspension of cells, 37% and 70% percoll are ready for use.

 

(6) Pipette 4mL of 30% Percoll, slowly enter a 15mL centrifuge tube along the tube wall, and cover the top of the 37% Percoll cell solution to form a density gradient;

 

(7) 900xg, 20 ℃, centrifugation for 45min, speed up (ACC) 1, speed down (DEC) 1;

 

(8) After centrifugation, draw the middle 37% and 70% Percoll interface layer cells (rich in TC) into a new 15mL centrifuge tube, add 3 times the volume of 4 °C pre-cooled 1xPBS, mix upside down, and centrifuge at 400g at 4 °C for 5min, discard the supernatant;

 

(9) Repeat the washing according to step (8), and discard the supernatant to obtain cells.

 

3. Cultivation and passaging

 

(1) Medium: DMEM + 10% FBS + 1% double antibody.

(2) Change the fluid every 2-3 days; When the cells reached 80% confluence, they were digested with 0.25% trypsin-EDTA for 1-2 min and passaged at 1: 3.

(3) The function is the most stable within the first 3 generations. It is recommended that the experiment be completed within this window.

 

4. Matters needing attention

 

1) The digestion time should not exceed 90min, so as not to reduce the viability rate.

2) When collecting cells after Percoll centrifugation, the interface layer needs to be accurately sucked to avoid mixing red blood cells or granulosa cells.

 

That's all for today's explanation. If you have any cell culture questions, please feel free to communicate with you!

 

Recommended by Xiaoai in this issue

Item number	Product name	Specifications
abs9483	DMEM basal medium	500mL
abs972	Fetal bovine serum	500mL
abs9244	Double antibody	100mL
abs9482	Tissue dissociation fluid	10mL
abs47014938	0.25% Trypsin-EDTA	500mL
abs961	10xPBS	500mL
abs962	1xPBS	500mL
abs9102	Percoll	100mL
abs7004	Cell culture dish (35mm)	1 box
abs7005	Cell culture dish (60mm)	1 box
abs7232	70um cell screen	1 box

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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