Issues with Low Purity in NK Cell Isolation from Cord Blood Using Miltenyi MACS Negative Selection Kit?

17 May 2024 0 8K Report

Hi everyone,

I am currently working on isolating NK cells from cord blood using the Miltenyi Biotec MACS negative selection kit. Despite following the protocol provided in the manual, I consistently end up with low purity in my NK cell populations. I would appreciate any insights or advice on potential reasons for this issue and any tips for improving the purity of the isolated NK cells.

Here is a summary of my procedure:

Collection and Initial Preparation:Collect cord blood in anticoagulant-treated blood bags from the obstetrics department of our collaborating hospital.

Density Gradient Centrifugation:Dilute the cord blood and layer it over Ficoll-Paque. Centrifuge at 450g for 30 minutes with an acceleration and deceleration setting of 1. Carefully collect the mononuclear cell layer (CBMCs).

Red Blood Cell Lysis:Treat the collected CBMCs with red blood cell lysis buffer according to the manufacturer's instructions. Incubate at room temperature for 1-5 minutes with gentle mixing. Add PBS or complete medium to stop the lysis reaction. Centrifuge at 1800 rpm to remove Ficoll. Centrifuge at 1200 rpm to remove platelets.

NK Cell Isolation:Proceed with the NK cell isolation using the MACS negative selection kit as per the provided protocol. I would greatly appreciate it if anyone could share their experiences and insights into the key steps that could impact the purity of NK cell isolation. Any specific tips for ensuring better purity in NK cell isolation from cord blood using this kit would be highly valuable.

Thank you in advance for your help!

Best regards,

Runjie Nie

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