XTT Assay: A Colorimetric Assay to Assess Cell Viability and Mitochondrial Activity

Overview

This article describes the protocol for XTT assay which is a colorimetric assay to assess cell viability and mitochondrial activity.

Protocol

1. XTT Assay to Determine Cell Viability and Mitochondrial Activity of Treated U937 and THP1 Cells

1. Prior to the XTT assays, grow U937 and THP1 cells under the same conditions. Pretreat cells with the same concentration range of MeβCD for 30 min prior to sonication. Use the same ultrasound parameters as before except that cells are now sonicated using a range of 1-3 one sec pulses spaced one sec apart to develop a range of damage for the XTT kit to assess.
   NOTE: Many of these steps are taken directly from the XTT kit manual and are only repeated for the convenience of interested laboratories.
2. Collect cells by centrifugation at 200 x g for 10 min. Resuspend cell pellet in the growth medium previously described. Resuspend cells at ~1 x 10⁵ cells/ml. Seed U937 cells at 100 µl per well into a flat-bottom 96-well microtiter plate in triplicate, and then repeat for THP1 cells using a separate 96-well plate. Include 3 control wells containing 100 µl of complete growth medium alone as blank absorbance readings. Then, incubate the inoculated plates for 24 hr.
3. Defrost two aliquots of the XTT reagent and the activation reagent at 37 °C prior to use. Swirl aliquots gently until clear solutions are obtained. Add 0.1 ml of the activation reagent to 5.0 ml of the XTT reagent, which forms enough activated XTT solution for one 96-well microtiter plate assay. Repeat the process for the other plate.
4. Add 50 µl of the activated-XTT solution to each well. Return the plate to the cell culture CO₂ incubator for 2 hr. Shake the plate gently following the incubation period to evenly distribute the orange color in the positive wells. Measure the absorbance of the assay wells containing the cells and the blank background control wells at a wavelength between 450-500 nm wavelength using a microtiter plate reader.
5. Either zero the microtiter plate reader using the blank control wells or subtract their average value from the specific results. Measure the absorbance of all the assay wells again at a wavelength between 630-690 nm and subtract the values from the 450-500 nm values obtained. Note: This second absorbance determination helps eliminate non-specific readings from the assay results. The XTT assay was repeated four times for both cell lines.

Materials

Name	Company	Catalog Number	Comments
Iscove's Modified Dulbecco's Medium  w/ NaHCO3 & 25mM Hepes	Life Technologies	12440079
Branson SLPe 40kHz Cell Disruptor with  3" (25mm) Cuphorn	Branson Ultrasonics	101-063-726	sonication device
Tip One 100 μl and 1,000 μl Filter Tips	USA Scientific	1120-1840, 1126-7810
100 μl Micropipette	Wheaton	851164
1000 μl Micropipette	Wheaton	851168
Forma Scientific Dual chamber water jacketed Incubator	Forma Scientific	3131
XTT Cell Proliferation Assay Kit	ATCC	30-1011K
96-Well Microplate Reader	Cole-Palmer	EW-13055-54
U937 Human Monocytic Leukemia Cells	ATCC	CRL1593.2
THP1 Human Monocytic Leukemia Cells	ATCC	TIB-202