Disgrego las médulas guardadas en Tissue Storage Solution el día anterior (sacadas de embriones E16 de rata.
Uso un kit que me deja Xavi: Adult brain dissociation kit (130-107-677, Milteny), y uso la máquina GentleMACS.
Protocolo kit:
Reagent and instrument preparation
▲ For subsequent cultivation it is recommended to dissociate at least 800 mg of adult neural tissue.
▲ Volumes given below are for one adult mouse brain (max. 500 mg) in 1980 μL enzyme mix. When working with less than 500 mg, use the same volumes as indicated. When working with neural tissue from adult rats or distinct brain regions, determine the weight and scale up all reagent volumes and total volumes accordingly. A maximum of 500 mg neural tissue per C Tube can be processed.
1. Enzyme P is ready to use. Prepare aliquots of appropriate volume to avoid repeated freeze-thaw-cycles. Store aliquots at –20 °C. This solution is stable for 6 months. Resuspend the lyophilized powder in the vial labeled Enzyme A with 1 mL Buffer A. Do not vortex. This solution should then be aliquoted and stored at –20 °C for later use. Avoid repeated freeze-thaw-cycles: en este caso las enzimas ya estaban alicuotadas.
2. Prepare enzyme mix 1 and enzyme mix 2 according to the table below (las enzimas están a -20ºC y los buffers a 4ºC)
- Enzyme mix 1: 50 μL Enzyme P + 1900 μL Buffer Z
- Enzyme mix 2: 20 μL Buffer Y + 10 μL Enzyme A
Dissociation protocol
▲ 20 mg up to 500 mg of rodent neural tissue in 2 mL enzyme mix can be processed in one C Tube.
▲ For cell culture experiments subsequent to tissue dissociation, all steps should be performed under sterile conditions.
1. Remove the rodent neural tissue. Wash neural tissue in cold D-PBS.
2. Prepare the appropriate volume of enzyme mix 1 (refer to table in section 1) and transfer it into a gentleMACS C Tube.
3. Cut larger tissue (e.g. whole brain or spinal cord) into approximately 8 sagittal slices or 0.5 cm pieces using a scalpel. In case of smaller tissue, e.g., hippocampus continue directly with step 4: en este caso no corto las médulas porque ya son suficientemente pequeñas.
4. Transfer the tissue pieces into the C Tube containing 1950 μL of enzyme mix 1.
5. Transfer 30 μL of enzyme mix 2 into the C Tube.
6. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Octo Dissociator with Heaters.
7. Run the appropriate gentleMACS Program:
- 20–100 mg: 37C_ABDK_02
- >100 mg: 37C_ABDK_01.
*Uso el programa 37C_ABDK_02 porque no hay mucha cantidad: poner el tubo boca abajo en el GentleMACS con la parte plana hacia adelante, poner el Heater (pieza amarilla), seleccionar programa (dura 30min).
8. After termination of the program, detach C Tube from the gentleMACS Octo Dissociator with Heaters.
9. Centrifuge briefly to collect the sample at the bottom of the tube.
10. Resuspend sample and apply it to a MACS SmartStrainer (70 μm) placed on a 50 mL tube.
▲ Note: Moisten MACS SmartStrainer with buffer before use.
▲ Note: When upscaling the reagent volume and total volumes, increase also the number of MACS SmartStrainers (70 μm). One MACS SmartStrainer (70 μm) can be used for one adult mouse brain.
▲ Note: Cells with a diameter >70 μm may be lost. To obtain these cells within the flow through, use a cell strainer with an appropriate mesh size.
11. Add 10 mL of cold D-PBS to the C Tube, close C Tube, shake gently and apply the D-PBS onto the MACS SmartStrainer (70 μm).
12. Discard MACS SmartStrainer (70 μm) and centrifuge cell suspension at 300×g for 10 minutes at 4 °C. Aspirate supernatant completely: en este caso no lo he centrifugado a 4ºC porque la centrífuga de cultivos no tieneregulador de temperatura.
13. Proceed to 2.3 for debris removal: no hago este paso!!!
*Después de aspirar el sobrenadante, resuspendo las células en 1 ml de medio C (co-culture médium), y las planto en 2 placas de 60cc con 26 cubres 9mm (previamente he hecho coating con Poly-D 1/40 overnight, y laminina 2’5 ul/ml 2h):
- DMEM (con D-glucosa y Glutamax): 44,5 ml (ref. 10569-010 ThermoFisher)
- 10% FBS: 5 ml
- 1% Pen-Str: 0,5ml
- 50 ng/ml NGF: 25 ul
