Protocols in molecular biology
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Toxicity or cell viability assays
September 7, 2019 at 5:23 pm #1353
Write or share protocols on in vitro toxicity or cell viability assays, including antimicrobial assays. Drug toxicology protocols are also appropriate. Please share links or provide article references. You may want to also attach published articles. Thank you for contributing to this topicSeptember 18, 2019 at 6:44 pm #1699
By Berthony Deslouches, MD, PhD
Red blood cell lysis assay
Mature red blood cells (RBC) or erythrocytes are non-nucleated cells that harbor hemoglobin in the blood. When red blood cells are lysed, they release hemoglobin, which can be measured spectrophotometrically at a wavelength of 570nm. The maximum percent of RBCs used in the assay (e.g., 2.5% RBC) would correspond to 100% hemoglobin released or 100% RBC lysis. An important consideration is the maximum amount of hemoglobin that can be measured in a plate reader without error. Commonly 2.5% is a good start, although this may depend on the instrument used. To calculate the percent RBC lysis, some investigators may be satisfied with just 100% and 0% lysis as control. Because of the potential for pipetting error, I recommend the use of a standard curve to analyze the test samples (Link to Deslouches et al. 2013) rather than just these two controls1. While RBC lysis in PBC demonstrates a direct lytic property of a drug, using whole blood to demonstrate lytic properties maybe more physiologically relevant, particularly n the context of systemic drug delivery2.
The following is a systematic description of the method, an example of standard curve, and calculation of the percent RBC in your test samples. Please feel free to share any comment, suggestion, or question.
The RBC lysis assay can be used to determine the hemolytic property of a drug.
• Whole blood or red blood cells already isolated, histopaque, DMEM ± NEAA (1%), V- or U-bottom + flat-bottom (for OD570 reading) plates
• drugs: 1mM or higher
1. Prepare drug samples (100uL after serial dilutions, in 96-well plates) at 2x maximum test concentrations in PBS (e.g., 2 x 1x will be 64, 32, 16, 8, etc.)
2. Blood (ignore this part if RBCs are already isolated):
• For preparation of PBMCs: separate PBMCs from RBCs by adding equal volume of whole blood (or buffy coat) to histopaque. Centrifuge @ ≥1000g/30min.
For RBC lysis assay
o Give RBC a hard spin and remove supernatant.
o If working RBC solution is too concentrated (reading is above capacity of the plate reader), then the plate reader will give an error. Usually 2.5-5% should be ok. I prefer 2.5% because it is below the maximum RBC that can be read by most plate readers without an error (too much too concentrated to read). Using too much RBC may also mask the RBC lytic potential of a drug when it is translated into percent lysis.
o Test for maximum reading at 570 nm in the plate reader: give the RBCs a hard spin and remove supernatant (PBS). Using 3 1.5mL tubes, add 1mL of dH20. Then add 20 (2%), 25 (2.5%), 50 (5%) µL RBC and shake well. The RBCs should lyse almost instantly (clear red, homogeneous solution with no pellet). Remove 200µL and add in triplicates using a 96-well plate. Read at 570nm. Choose 2.5% RBC if the absorbance is obtained without error as maximum RBC concentration.
o RBC: final concentration 2.5 to 5%, make a 2x RBC in PBS
o Standard curve: from final 1x RBC concentration, transfer 50uL, 45, 40, 35, 30, 25, 20, 15, 10, and 5uL into d-water: 450uL, 455, 460, 465, 470, 475, 480, 485, 490, 495, and 500uL (The RBC is diluted to maximum 10% and lysed).
o Prepare your peptides in a U-bottom 96-well plate at 2x concentrations
o Transfer 100uL 2x RBC to the 2x peptide solutions
o Shake well and Transfer the appropriate volume of standard to one raw of wells containing d-water to obtain the desired final dilution [e.g., from 10% to 5% (100uL) or 2.5% (50uL)]
o Incubate at 37deg C for 60min
o Spin plate at 500-1000g for 5-7min
o Using a flat-bottom well, Add 100uL supernatant from each peptide-RBC mixture to each corresponding well (make sure to avoid contact with RBC pellet) and transfer 100uL of all standards to the plate.
o Read at 570nm in a plate reader
o All done!
Example of a standard curve is attached. The total amount of RBCs used was 2.5% RBC representing 100% lysis at A570: in the equation Y=0.013x + 0.1023, replace Y with the absorbance of your test sample to find X as %RBC lysis. Credit: Lab Technicians who performed this experiment, Wenyu Xiang, Patrice Clemenza, and Jessie Klousnitzer.
1 Deslouches, B. et al. Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens. Antimicrob Agents Chemother 57, 2511-2521, doi:10.1128/AAC.02218-12 (2013).
2 Deslouches, B. et al. Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications. Antimicrob Agents Chemother 49, 3208-3216, doi:10.1128/AAC.49.8.3208-3216.2005 (2005).
- This reply was modified 1 day, 14 hours ago by Nicole Stewart.
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