I am following step by step a published protocol but I am having some problems to isolate Sertoli cells. See attached a picture of Day 1 culture. Could anyone with experience help me to understand if I am doing something wrong?
This is the protocol
Sertoli Cell Culture (Oliveira, Sousa et al. 2009)
1) Transfer testes to cold calcium- and magnesium- free Hanks balanced salt solution (HBSSf) containing 50 U/ml of penicillin and 50 µg/ml of streptomycin sulphate (pH 7.4) immediately after removal.
2) Decapsulate tissue (2 g) and wash twice and cut into small square pieces (2–4 mm) in a sterile Petri dish
3) Suspend minced tissue in HBSSf (25 ml/g of tissue) in a glass-stoppered 100-ml Erlenmeyer and shake vigorously for 1 min to disperse tubules.
4) Leave the tissue to seattle for 5 min on ice, and discard the supernatant.
5) Repeat this procedure twice to mechanically remove red blood cells and free Leydig cells.
6) Digest the resulting pellet in 25 ml of HBSS with collagenase type I (1,000 U; C0130, Sigma) and DNAse (500 U; D4263, Sigma) and continuously shake (100 rpm) at 32ºC for 25– 35 min.
7) Remove the formed aggregate, wash in HBSSf and discard. Add the washing HBSSf to the cellular suspension resulting from the digestion.
8) Wash the resulting suspension twice and leave to settle completely at 4ºC
9) Suspend the resulting pellet in 20 ml HBSSf with 5 mg pancreatin (P3292, Sigma) and DNAse (500 U, D4263) and digest at 32ºC with continuous shaking (100 rpm) for 15–25 min.
10) Discard the new aggregate formed, and add 0.4 ml of fetal bovine serum to the cellular suspension, which has left to rest at 4ºC for 5 min
11) Centrifuge the suspension at 100g for 5 min.
12) Gently suspend the pellet in 30 ml HBSSf. Repeat this procedure twice, and suspend the resulting pellet in 20 ml HBSSf.
13) Pass this suspension through a glass Pasteur pipette in order to loose germ cells from the clusters and then pellet at 200g for 5 min. repeat this procedure twice.
14) Suspend the resulting pellet 10 ml Sertoli culture medium (DMEM-Ham’s F-12 [HF12];
1:1, containing 50 U/ml of penicillin and 50 µg/ml of streptomycin sulfate, 0.5 µg/ml fungizone and 5% heat inactivated fetal bovine serum) and force through a 19G needle in order to disaggregate large Sertoli clusters.
15) For culture of Sertoli cells, the concentration of clusters on the cellular suspension obtained from the procedure described above has to adjusted to 1,000 clusters/ml plated on 25 cm2 culture flasks (Cell; Sarstedt, Leicester, UK) and incubated at 37ºC in an atmosphere of 5% CO2:95% O2. The day of plating was considered day 0 of culture. Cultures were left undisturbed until day 2.
References
Oliveira, P. F., M. Sousa, et al. (2009). "Membrane Transporters and Cytoplasmatic pH Regulation on Bovine Sertoli Cells." Journal of Membrane Biology 227(1): 49-55.
Refer this paper
Effect of argemone oil and argemone alkaloid, sanguinarine on Sertoli-germ cell coculture. Toxicology Letters 05/2009; 186(2):104-10.
This paper is from my previous lab where one of my senior was involved in this work. Try to follow the protocol given in this paper. May be your problem will be solved
I can suggest you a pretty much easier protocol. You can see this protocol here in my recently published paper:
http://link.springer.com/article/10.1007%2Fs11626-014-9762-1
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