Primary culture of lymphocytes and monocytes

DATA:
Start of the experiment: Day 15-05-2023
End of the experiment: Day 16-05-2023

OBJECTIVE:
Aprender to obtain a primary culture, of lymphocytes and monocytes, from human
blood and cultivate them under in vitro conditions.

MATERIAL:
PBS, Medi RIPA + 1%P/s (15140-122) + 10% FBS(11573397), flask roux, Whole blood,
p100, Needle, EDTA blood collection tube, Ficoll.

METHOD:

blood draw
Centrifuge at 400 g, 20 min, room T°.
In a 15 ml falcon add 5 mL of PBS and 5 mL of whole blood, in another 15 mL falcon
we add 3 mL of ficoll and add the mixture of PBS and whole blood, and finally
centrifuge 400 g, 20 min, ambient temperature

1. Very carefully remove the yellow layer of serum.

2. Carefully collect the layer of PBMCs in a P100 and transfer to a 15 mL falcon.
 3. Suspend with 10 ml of PBS (room temperature) and mix gently.
4. Centrifuge at 100 g, 10 min, room temp.
5. Remove SN and repeat steps 10 and 11, then remove SN and reconstitute
cells in 1 mL of medium.
6. We seeded 10 mL in a P100 and labeled monocytes.
7. Incubate at 37ºC, 5% CO2 for at least 1h.
8. We collect the medium from the P100 and put it into a 50 mL falcon.
9. Let's seed 2 roux flasks with the medium collected from the P100 where the
lymphocytes will be found.

RESULTS AND CONCLUSIONS:

1. The project had problems centrifuging whole blood where the layer of PBMCs
could not be identified.
2. The option of collecting and seeding the plasma phase was taken to be able
to observe if the lymphocytes and monocytes were found.
3. When looking at the seeded plates under a microscope, it was possible to
observe various organisms that could be both lymphocytes, monocytes,
platelets...