Isolating PBMCs from frozen buffy coat samples without a cryoprotectant is challenging because cell membranes often rupture during the freeze–thaw cycle, leading to poor viability. However, depending on your end goal (viable cells vs. protein recovery), there are some options you could try:

If you aim to recover PBMCs (viable or not):

• Expect low cell viability, but you can still isolate some intact cells using Ficoll or Lymphoprep gradients.
• Thaw the samples quickly at 37°C, dilute slowly with cold PBS or RPMI to minimize osmotic shock.
• Add DNase I (e.g., 100 U/mL) during the wash step to reduce clumping from lysed cells.
• Perform cell counting with trypan blue to assess recovery.

If you're focused on protein recovery:

• Skip PBMC isolation and proceed directly with cell lysis using RIPA buffer or other appropriate buffers for your target proteins.
• Centrifuge to remove debris and quantify protein with a BCA or Bradford assay.
• Consider adding protease/phosphatase inhibitors during lysis to preserve protein integrity.

Unfortunately, PBMC viability will likely be severely compromised, but total protein extraction is usually feasible.

Let me know if you're targeting specific markers or downstream applications like Western blot or ELISA—I’d be happy to suggest more tailored protocols