Fast response temperature measurement and highly reproducible heating methods for 96-well plates

Abstract

Hyperthermia, the procedure of exposing cells to a temperature between 42 degrees and 49 degrees C, has been shown to be a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, it is critical to have an accurate temperature measurement technique and a heating method with high reproducibility. To this end, we have developed a method using fine thermocouples with fast response time to measure the temperatures in multiple wells of a 96-well plate. The accuracy of temperature measurement was +/- 0.2 degree C. Such a capability allows a complete record of the time and temperature of the treatment procedure and helps define an accurate thermal dose. We have also compared several methods for heating 96-well plates and found that use of copper blocks in contact with the lower surface of the 96-well plate in an incubator provides a highly reproducible heating method. The common method of using water bath to heat cells in vitro resulted in a decrease of cell viability even at the control temperature of 37 degrees C and a decrease in the reproducibility of certain biological assays. In summary, using these improved techniques, proposed thermal dose can be defined more precisely, and highly reproducible heating in vitro can be achieved.

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection

“…We have shown for the first time that NKI‐beteb was expressed specifically in melanocyte lineage cells in the normal hair follicle bulge (Fig. S2b,d), offering additional information to other studies that described NKI‐beteb expression in the amelanotic and pigmented melanocytes in the hair follicle outer root sheath . Further, we have shown that the NKI‐beteb antibody is an excellent tool for identifying a wide range of melanocytes in various stages of differentiation from hair follicles and epidermis.…”

“…S2b,d), offering additional information to other studies that described NKI-beteb expression in the amelanotic and pigmented melanocytes in the hair follicle outer root sheath. 9,24 Further, we have shown that the NKI-beteb antibody is an excellent tool for identifying a wide range of melanocytes in various stages of differentiation from hair follicles and epidermis. Moreover, the NKI-beteb antibody exhibited strong signal intensity in frozen tissue samples and in samples subjected to our rapid staining protocol, making this antibody a useful tool for minimizing RNA degradation.…”

“…F‐LCM was performed on slides within 1 hour of completing immunostaining. We used an infrared laser (Arcturus XTTM) microdissection system, under direct fluorescent microscopic visualization, in the LCM Core at the University of Colorado, using cold copper blocks and standard techniques detailed in the Supplementary Materials and Methods. From each sample, we captured 100 NKI‐beteb‐positive melanocytes from the epidermis, 100 NKI‐beteb‐positive cells from the hair follicle bulge (Fig.…”

Hyperthermia Induces Endoplasmic Reticulum-Mediated Apoptosis in Melanoma and Non-Melanoma Skin Cancer Cells

“…Cells were treated using methods we described previously (Shellman et al, 2004). Briefly, cells were grown in 96-well plates with 100 ml medium using standard culture conditions overnight before treatment.…”

Development of Highly Sensitive Temperature Microsensors for Localized Measurements

“…This measurement must be effective and accurate, however, when the samples are of micro-or even of nano-order, as in OoC, this task becomes quite difficult [12]. Several approaches for cell culture temperature sensing have been explored in previous works such as commercial T-type thermocouples [13], NTC (negative temperature coefficient) thermistors [14], commercial PT-100 RTDs (resistance temperature detectors) [15], fluorescent polymeric thermometers [16] and optoacoustic methods [17]. However, conventional sensing devices for controlling the cellular microenvironment are not very compact for OoC applications and use expensive and bulky instrumentation equipment [11,18].…”