RECOMMENDED SAMPLE HANDLING AND PROCESSING

Blood, Plasma, and Serum

General blood sample processing requirements

Proper processing of the collected samples is critical. It is particularly important that time constraints are observed and that samples are not left at room temperature longer than necessary. Samples should be processed and frozen at ‐80 °C within 2 hours of collection.

Plasma

Centrifuge plasma tubes (Citrate, Heparin or EDTA tubes) at room temperature. If within tube manufacture’s specifications, spin at 2200 x g (not RPM) for 15 minutes (this speed has been chosen to attempt to remove all cellular contents and platelets from samples). Observe separation of blood cells and plasma, with plasma layer on top.  Draw off only the plasma layer. Take care not to disturb buffy coat when aliquoting by leaving some plasma behind and avoiding the cell layer. Aliquot into appropriately labeled tubes. Aliquot samples immediately and then place aliquoted samples in a ‐80 °C freezer.

Note: Plasma samples do not need to clot, and should be centrifuged immediately after collection.

Serum

Allow serum to clot for 60 minutes at room temperature prior to centrifugation. Centrifuge serum tubes. If within tube manufacture’s specifications, spin at 2200 x g (not RPM) for 15 minutes (this speed has been chosen to attempt to remove all cellular contents and platelets from samples). Observe separation of blood cells and serum, with serum layer on top. Draw off only the serum and aliquot into appropriately labeled tubes. Aliquot samples immediately and then place aliquoted samples in a ‐80 °C freezer.

Urine

Collect neat urine sample. Clarify the urine by centrifugation at 14,000 x g for 5 minutes, prior to freezing, and collect the clarified supernatant. Store aliquots at ‐80 °C.

Cell culture lysate 


Collect samples using Mammalian Protein Extraction Reagent (Thermo Scientific) following manufacturer instructions. Sufficient material can usually be obtained from a cell monolayer, 80-100% confluent, in a single well of a six‐well plate, harvested with 300 μL lysis buffer.

To harvest cell lysate: -Wash cells three (3) times with Dulbecco’s Phosphate Buffered Saline (DPBS) prior to lysing. Add protease inhibitor cocktail to the lysis buffer to inhibit protease activity. Add lysis buffer to the cells followed by appropriate lysis procedure. Centrifuge lysed cells at 14,000 x g for 5 minutes, and collect the supernatant (clarified lysate). Quantify total protein amount using BCA Protein Assay Kit or similar protein quantification method.

Cell conditioned media (Cell culture supernatants)

For studies with cells cultured in 1‐10% fetal bovine serum, consider including proper control samples (media controls, untreated cells and/or vehicle‐treated cells) depending on the scientific question to be addressed. Keep the media volume to a minimum in order to increase protein concentration, and strive to have cell density at 75% surface area or greater. Sufficient material can usually be obtained from 1 mL media removed from 80‐100% confluent cell monolayer from a single well of a six‐well plate. Time points of less than 24 hours may be too early to show a differential signal, consider reducing the volume of media used for these types of experiments. Clarify cell supernatant by centrifugation at 14,000 x g for 5 minutes, prior to freezing, and collect the clarified supernatant. The minimum volume required of clarified supernatant is 100 μL. Store samples in a ‐80 °C freezer.

Tissue or xenograft tumor homogenates

Snap freeze (at least 5 mg) excised tissue in liquid nitrogen within 5‐10 minutes of excision. Pulverize frozen tissue (using a freezer mill or similar) while maintaining low temperature using liquid nitrogen or dry ice. Use T-Per tissue protein extraction agent (Thermo Scientific) per manufacturer’s recommendation. Add 200 uL extraction buffer plus protease inhibitor cocktail per 10 mg of tissue. Homogenize in tube on ice with rotary pestle for 30 seconds, until no tissue fragments are visible.  Centrifuge while cold at 14,000 x g for 10 minutes. • Collect supernatant (keep on ice). • Filter through a 0.2 micron filter into a sterile tube or plate. • Quantify total protein amount using BCA Protein Assay Kit or other similar protein quantification method.

Sample Size Requirements

Depends on the analyte to be assayed. Please contact techsupport@novateinbio.com.