Strategy for extracting rat ovarian theca cells

1. Preparation before the experiment

 

1) Animals:

SD rats (weight 180-220g) with regular sexual cycle of 6-8 weeks.

 

2) Main reagents

DMEM/F12 basal medium (abs9560)

Fetal bovine serum (abs972)

ITS (abs9462)

Double antibody (abs9244)

Tissue dissociation solution (abs9482)

0.25% Trypsin-EDTA (abs47014938)

10xPBS (abs961)

1xPBS (abs962)

Percoll (abs9102)

70um cell screen (abs7232)

Cell culture dish (35mm) (abs7004)

Cell culture dish (60mm) (abs7005)

1 mL Injection needle

Antibodies: CYP17A1 (rabbit polyclonal antibody, labeled TC) and FSHR (rabbit polyclonal antibody, granulosa cells excluded)

 

2. Extraction and sorting of theca cells

 

01 Ovarian sampling

 

Cervical dislocation sacrifice → 75% ethanol disinfection → rapid removal of bilateral ovaries → placement in ice DMEM/F12 (+1% double antibody).

 

02 Follicular preconditioning

 

(1) Remove the ovarian capsule and corpus luteum with microscopic forceps → Place the ovaries in a 35mm dish and add 1mL DMEM/F12.

(2) Repeatedly puncture the follicle with a 1mL injection needle, release and discard the granulosa cell-oocyte complex, and retain the residual wall of the follicle (rich in membrane cells).

 

03 Enzymatic digestion

 

(1) Cut the residual wall of the follicle into small tissue blocks of 0.5-1mm3, transfer the tissue blocks (100-200mg) to a 2mL round-bottomed tube, add 1mL digestive juice (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, scroll the tube vigorously for 10-20s, filter through a 70um cell sieve (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.

 

04 Percoll density gradient purification

 

(1) 35% 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 35% isotonic Percoll solution was prepared by mixing 100% Percoll mother liquor and 1 × PBS at a volume ratio of 7:13.

 

(2) 50% Percoll solution

Percoll cell separation solution was mixed with PBS buffer (10x) at a volume ratio of 9: 1 to prepare isotonic 100% Percoll mother liquor Mix 100% Percoll mother liquor and 1x PBS at a volume ratio of 1: 1 to prepare 50% isotonic Percoll solution

 

(3) Pipette 1mL of 50% percoll deep into the bottom of 15mL centrifuge tube

 

(4) Resuspend that cell pellet with 2mL, 35% percoll (abs9102), slowly add it to a centrifuge tube containing 1mL of 50% percoll along the tube wall, and cover the top of the 50% Percol solution to form a density gradient

▲ Note: Do not destroy the interface between 35% percoll and 50% percoll and the suspension of cells, 35% and 50% percoll are ready for use

 

(5) 400xg, 20 ℃, centrifugation for 10 min, speed up (ACC) 1, speed down (DEC) 1

 

(6) After centrifugation, draw the middle 35%/50% interface layer cells (rich in TC) into a new 15mL centrifuge tube, add 3 times the volume of 4 ℃ pre-cooled 1xPBS, mix upside down, and centrifuge 400g at 4 ℃ for 5min, discard the supernatant

 

(7) Repeat washing once according to step (6), discard the supernatant to obtain cells

 

05 Differential sticking to the wall to further remove impurities

 

(1) Resuspend the cells in DMEM/F12 + 10% FBS + 1% double antibody, inoculate on a 6cm dish, and incubate at 37 °C and 5% CO ₂ for 2h.

(2) Gently aspirate the unadherent granulosa cells, and change them to fresh culture medium to continue culture.

 

06 Purity identification

 

(1) Take cells cultured for 24 hours for immunofluorescence: CYP17 A1 ⁺/FSHR ⁻ determined to be TC, and the purity is usually > 90%.

(2) qPCR can also be used to detect the high expression of Lhcgr and Cyp17a1, but the low expression of Fshr.

 

3. Cultivation and passaging

 

01 Medium: DMEM/F12 + 10% FBS + 1% ITS + 1% double antibody.

 

02 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 1: 3.

 

03 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

 

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

 

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

 

03 If serum-free experiments are required, FBS can be reduced to 2% and ITS (insulin-transferrin-selenium) can be added.

 

Recommended by Xiaoai in this issue

Item number	Product name	Specifications
abs9560	DMEM/F12 Basal Medium	500mL
abs972	Fetal bovine serum	500mL
abs9462	ITS	10mL
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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