Flx800 fluorescence reader

The resazurin reduction test was used to measure cell viability as previously described (O'Brien, Wilson, Orton, & Pognan, 2000). Briefly, 0.1 mm resazurin solution salt solution in basal culture media was added to each sample and incubated for in the dark for 4 h at 37 °C; 200 μL of pink resorufin product was transferred to a microtitre plate, and measured spectrofluorometically (FLx800 Fluorescence Reader; BioTek, Potten, UK) at an excitation wavelength of 540/35 nm, and emission wavelength of 630/32 nm.

Nuclear extracts were prepared by using the NE-PER® nuclear and cytoplasmic extraction reagents (Pierce) as per the manufacturer’s instructions. The DNA binding assay was performed as described [29 (link)] with some modifications. Briefly, 5~10 μg of nuclear extracts were mixed with poly dI-dC (50 μg/ml) in a binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, 3 mM MgCl₂, 5 % glycerol, 0.5 mg/ml BSA, 0.05 % NP-40) and then incubated in 96-well plates coated with immobilized biotin-labeled oligonucleotides (2 pmol per/well) for 1 h at room temperature. Following three washes, primary antibody specific to NF-κB p65 was added and incubated again at room temperature for 1 h. Addition of secondary antibody conjugated to horseradish peroxidase was performed prior to the quantification of NF-κB DNA-binding activity by measuring luminescence (FLx800 Fluorescence Reader, Biotek Instruments, Inc., Winooski, VT). The following biotin-labeled oligonucleotides were used: the consensus NF-κB, 5′-CACAGTTG- AGGGGACTTTCCCAGGC-3′; the four putative NF-κB sequences within the mouse TNF-α promoter, κB1 (−856): 5′-GGGGGAGGGGAATCCTTGGAAGAC-3′; κB2 (−659): 5′-GAG- GTCCGTGAATTCCCAGGGCTG-3′; κB3 (−512): 5′-CAAACAGGGGGCTTTCCCTCCT-CA-3′; κB4 (−214): 5′-GACGGGGAGGAGATTCCTTGATGC-3′.

Msm liquid cultures were induced with 0.2% acetamide in early log phase (OD_590nm = 0.5) and samples were collected every two hours. Fluorescence at 525 nm (excitation at 485 nm) and OD_590nm were measured in micro-titration plates (Optiplate-96 well) with a FLx800 fluorescence reader (Biotek Instruments). Acquisition of fluorescence in the sample (F(s)) was performed in triplicate on serial dilutions of each culture. The data, expressed in relative fluorescence units (RLU), were corrected for the variation in fluorescence of the medium (F(m)) and the background fluorescence of the Msm/pLAM12 control strain (F(c)). Fluorescence (F) was also reported to the OD of the sample (OD(s)) and of the control strain (OD(c)) using the following formula: F  =  [(F(s) – F(m))/OD(s))]/[(F(c) – F(m))/(OD(c)]. The means ± SD (standard deviation) of the data were plotted against the time of induction using Graphpad Prism 5 software (GraphPad Software Inc.). The same culture samples were analyzed by Western blotting during the course of induction. Aliquots of known and equivalent OD were lysed, fractionated on 10% SDS-PAGE and appropriate amounts of total protein were blotted using mouse monoclonal anti-GFP (Roche Applied science ref: 11814460001) as primary antibodies. The blots were revealed by bioluminescence (ECL detection Kit, Millipore) and visualized by fluoroimaging.

Stro-1-enriched SSCs cultured in six-well plates were washed with PBS and fixed with 95% ethanol in dH₂O for 10–15 min. Cells were lysed using 0.05% (v/v) Triton™ X-100 solution in dH₂O followed by three freeze–thaw cycles. Total DNA was quantified against a standard curve (ssDNA from salmon testes) by addition of Quant-iT™ PicoGreen^® dsDNA diluted in TE buffer and measurement of the emitted fluorescence (λ_ex = 480 nm, λ_em = 520 nm) using a FLx800™ fluorescence reader (BioTek, Winooski, VT, US). ALP activity was quantified against a standard curve (p-nitrophenol) by incubation of the cell lysate with a phosphatase substrate solution (1.33 mg ml⁻¹p-nitrophenyl phosphate in 0.5 M alkaline buffer solution) at 37°C in the dark, under gentle agitation. The reaction was terminated by addition of 1 M NaOH in dH₂O after 40 min and absorbance was measured at 410 nm using an ELx800™ absorbance reader (BioTek) [22 (link),45 ].

Lipid peroxidation results in the formation of Malonaldehyde bis (MDA) and its level can be determined through interaction with thiobarbituric acid forming a fluorescent product [54 (link)]. Briefly, BM cells in sodium phosphate buffer (10 mM; PH 7.2) containing butylhydroxytoluene (BHT) (0.2 mM; in 0.15% ethanol solution) and EDTA (1 mM) were lysed through 3 cycles of freezing and thawing in liquid nitrogen. Thiobarbituric acid (0.5%) was added, the mixture was heated (100 °C; 15 min), and the fluorescent product was extracted into Butanol and measured (Ex. 485 nm/Em. 528 nm) using an FLx800 Fluorescence Reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The MDA results are presented as nmole/mg protein using Malonaldehyde bis + diethyl acetal as a standard curve. All reagents used in this assay purchased from Sigma (St. Louis, MO, USA).

Monarchs were collected as either larvae or adult butterflies from various locations across their current geographical range between 1990 and 2017 (Table S1). DNA was extracted from samples using a magnetic bead‐based protocol (Ali et al., 2016 (link)) and quantified using Quant‐iT PicoGreen dsDNA Reagent (Thermo Fisher Scientific) on an FLx800 Fluorescence Reader (BioTek Instruments). Restriction‐associated digest (RAD) DNA libraries were then created using the PstI restriction enzyme according to Ali et al. (2016 (link)) and sequenced using 150‐bp paired‐end sequencing on an Illumina Hi‐Seq 4000.