Hello Cleyson da Cruz Oliveira Barros

You may use the following protocol.

1. Transfer cord blood into a 50 mL conical tube and then dilute to 1:1 ratio with Hanks’ balanced salt solution (HBSS) (w/o Ca2+ and Mg2+).

2. This diluted blood is then gently layered on top of Ficoll at 2:1 ratio (two volumes of blood diluted with HBSS (w/o Ca2+ and Mg2+) to one volume Ficoll).

3. The conical tube is then centrifuged at 1000g for 30 min at room temperature with brake inactivated to allow layering of cellular components.

4. The cloudy ring below the plasma and above the Ficoll (i.e., the cord blood-MNC layer) is collected and placed in a new 15 mL conical tube, with HBSS (w/o Ca2+ and Mg2+) added to bring the volume to 15 mL.

5. This tube is centrifuged at 330g for 10 min with high brake.

6. The supernatant is removed and the cord blood-MNC pellet is then washed with HBSS (w/o Ca2+ and Mg2+) and resuspended in 10 mL HBSS (w/o Ca2+ and Mg2+) for counting.

7. You may count the cells on a hemocytometer in a 1:10 dilution of trypan blue.

If you wish to freeze the cells, you may follow the protocol as given below.

Cells may be frozen in freezing medium consisting of RPMI 1640 Medium with 1% penicillin–streptomycin, 2mM L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 20% fetal bovine serum (FBS), and 10% DMSO at 5–10 million cells/ vial, placed in a chilled Mr. Frosty freezing container for gradual freezing at -1 degree C/min and then place the freezing container into −80 °C freezer overnight. The following day, cord blood-MNC cryovials may be transferred to liquid nitrogen for long-term storage.

Best.

To isolate cells from human cord blood using Ficoll-Paque, you can follow these general steps:Gather Materials:Ficoll-Paque solutionCord blood samplePhosphate-buffered saline (PBS) without calcium and magnesiumCentrifuge tubesCentrifugePipettes and pipette tipsDilute Cord Blood:Dilute the cord blood sample with an equal volume of PBS without calcium and magnesium in a centrifuge tube.Layering on Ficoll-Paque:Carefully layer the diluted blood sample onto the Ficoll-Paque solution in a new centrifuge tube. Use caution to avoid disturbing the layers.Centrifugation:Centrifuge the tube at a specific gravity for a specific duration, as per the protocol you are following. This step separates the blood components based on their density, with the lighter cells moving towards the top and the heavier ones sinking to the bottom.Collecting Cells:After centrifugation, you will see distinct layers in the tube. The middle layer contains the mononuclear cells (including lymphocytes and monocytes). Carefully collect this layer using a pipette, being cautious not to disturb the layers above and below.Washing the Cells:Transfer the collected cells into a new centrifuge tube and wash them with PBS. Centrifuge the tube again to pellet the cells. Discard the supernatant and resuspend the cells in the desired medium for further analysis or experimentation.Counting and Viability Check:Count the isolated cells using a hemocytometer or an automated cell counter. Also, check the viability of the cells using a trypan blue exclusion assay or a similar method.