PBMC Isolation and Cryopreservation

Peripheral Blood Mononuclear Cells (PBMC) isolation and cryopreservation are key laboratory procedures in immunology.

Safety Precautions

Strict adherence to safety measures is critical to prevent biohazard exposure and cross-contamination.

Personal Protective Equipment (PPE)

Wear lab coat, gloves, and protective eyewear at all times in the lab.

Biological Safety Cabinet

Perform all procedures involving live cells within a certified biosafety cabinet.

Biohazard Disposal

Dispose of all biological waste properly in biohazard bins.

Chemical Handling

Follow Material Safety Data Sheet (MSDS) guidelines for chemical reagents handling.

Equipment Sterilization

Sterilize all equipment before and after use to maintain aseptic conditions.

Materials

Prepare and organize all necessary materials before starting the procedure.

Blood Collection Tubes

Use heparinized tubes or tubes with anticoagulants to prevent blood clotting.

Pipettes and Tips

Have a range of pipette sizes and sterile tips available.

Centrifuge Tubes

Ensure that sterile, appropriately sized centrifuge tubes are on hand.

Freezing Vials

Use cryovials designed for low-temperature storage.

Labels and Markers

Prepare labels for clear sample identification.

Equipment

Ensure all equipment is calibrated and functioning properly before use.

Centrifuge

Use a centrifuge capable of generating the required g-force.

Hemocytometer or Automated Cell Counter

For accurate cell counting before cryopreservation.

-80°C Freezer or Liquid Nitrogen Storage

For long-term storage of cryopreserved cells.

Biosafety Cabinet

Provides a sterile work environment for sample handling.

Reagent Preparation

All reagents should be prepared fresh or thawed properly prior to use.

PBS or Isotonic Buffer

For washing and resuspending cells during the procedure.

Density Gradient Media

For separation of PBMCs from other blood components.

Cryopreservation Media

A mixture typically containing FBS and DMSO to protect cells during freezing.

Alcohol Wipes

For cleaning surfaces and equipment.

Isolation Procedure

Follow a standardized protocol to ensure the purity and viability of PBMCs.

Blood Dilution

Mix blood with PBS or isotonic buffer prior to density gradient separation.

Density Gradient Centrifugation

Carefully layer diluted blood over density media and centrifuge to separate PBMCs.

PBMC Collection

Harvest the mononuclear cell layer without disturbing other layers.

Washing Steps

Wash collected PBMCs multiple times in buffer to remove residual media.

Resuspension

Resuspend PBMCs in an appropriate volume of cryopreservation media.

Cell Counting

Accurate cell counting is crucial for determining cell concentration before cryopreservation.

Sample Mixing

Mix a small aliquot of cell suspension with a trypan blue solution or equivalent.

Loading Hemocytometer

Fill the hemocytometer with the stained cell mixture for counting.

Viability Assessment

Calculate the percentage of live cells to ensure high viability.

Concentration Calculation

Determine the cells/mL concentration for cryopreservation aliquoting.

Cryopreservation

Proper cryopreservation is essential for maintaining cell function after thawing.

Aliquoting

Dispense cells into pre-labeled cryovials according to desired cell numbers.

Controlled Rate Freezing

Gradually decrease the temperature to prevent cell shock and ice crystal formation.

PBMC Isolation and Cryopreservation