A-431 cells were obtained from ATCC (Manassas, Virginia). Cetuximab (Bristol-Myers Squibb, Princeton, New Jersey) was conjugated according to the manufacturer’s instructions with each of the following dyes: CellTraceTM Far Red DDAO-SE (DDAO) (Life Technologies, Eugene, Oregon), IRDye® 800CW NHS Ester (IRDye)(LI-COR, Lincoln, Nebraska), Alexa Fluor® 680 NHS Ester (AF680) (Life Technologies, Eugene, Oregon), Alexa Fluor® 750 NHS Ester (AF750) (Life Technologies, Eugene, Oregon), Sulfo-Cyanine7 (SulfoCy7) (Lumiprobe Corporation, Hallandale Beach, Florida), Cy5.5 (GE Healthcare Bio-Sciences, Pittsburgh, Pennslyvania), Bodipy® 650/660-X (BODIPY-650) (Life Technologies, Eugene, Oregon), Atto 740 NHS Ester (Atto 740) (Sigma-Aldrich Corp., St. Louis, Missouri). Dyes were reacted in 10% sodium bicarbonate and antibody solution (2mg/mL) for 2 hours at room temperature. A molar ratio of 1.0 was used for all dyes. The antibody-dye conjugates were purified using 800uL of 5g/50mL water of Biogel P-6, Fine (Bio-Rad, Hercules, California) in Spin-X centrifuge filter tubes (Corning, Corning, New York). The final degree of labeling was determined by the absorption at 280nm corrected for the fluorophore and the max absorption wavelength of each dye using a NanoDrop 1000 spectrophotometer. A protein gel was run to ensure that there was no free dye remaining.
Spin x centrifuge filter tubes
Manufactured by Corning
Spin-X centrifuge filter tubes are a laboratory equipment designed for sample separation and filtration. They feature a unique centrifuge tube design with an integrated filter membrane that enables efficient separation of particulates, cells, or other suspended matter from liquid samples during centrifugation.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw nhs ester, by LI COR (1 mentions)
Cetuximab, by Bristol-Myers Squibb (1 mentions)
A431 cells, by American Type Culture Collection (1 mentions)
Centricon plus 70 filter unit, by Merck Group (1 mentions)
Lenti x 293t cells, by Takara Bio (1 mentions)
Ultraculture serum free media, by Lonza (1 mentions)
Transit reagent, by Mirus Bio (1 mentions)
4 protocols using spin x centrifuge filter tubes
1
Fluorescent Labeling of Cetuximab
2
Headspace Gas Analysis for Microbial Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Headspace O2 was determined by ECD-GC using a molecular sieve 5A column (3.2 mm O.D. × 2.4 m) operated at 75°C using hydrocarbon-free UHP N2 carrier. Background O2 was minimized by flushing syringes and needles with O2-free N2 prior to sampling. The detection limit was 0.05 mmol O2/L. Headspace CH4 and CO2 were determined by FID- and TCD-GC, respectively (Miller et al., 2013 (link)). Cell densities were determined by direct cell counting of liquid samples using acridine orange epi-fluorescence microscopy (Hobbie et al., 1977 (link)). Additional aqueous samples, including slurries, were filtered using Spin-X centrifuge filter tubes (0.2 μm; Corning Inc., Corning, NY) before determination of dissolved acetate by HPLC (Hoeft et al., 2004 (link)) or anions by IC (Miller et al., 2003 (link)). Dissolved ClO−4 was analyzed separately by suppressed conductivity IC using a Dionex ISC 1100 containing an AS16 analytical column (4 × 250 mm) and an AG16 guard column (4 × 50 mm) with 0.035 M NaOH eluent. Measurements of headspace 14CH4 and 14CO2 were made by gas proportional counting (Culbertson et al., 1981 (link)) following TCD-GC analysis of CH4 and CO2 with separation on a Hayesep D column (100/120; 3.2 mm O.D. × 4.8 m) using UHP He carrier.
3
Protein Extraction from Dried Blood Spots
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From each card, five 6.9 mm diameter disks were prepared using a hole punch and transferred into CoStar Spin-X Centrifuge Filter Tubes (7200388). Proteins were extracted from the DBS using 333 µL of elution buffer (M-PER with salt, Antipain, Chymostatin, Protease Inhibitor) with orbital shaking overnight at room temperature. The eluates were collected after a 6-min centrifugation at 12,000×g and immediately used in the assay. Fifty microliters of the eluate were used per well in the assay (Fig. 1 ).
4
Production of Replication-Incompetent Lentivirus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To produce live, replication-incompetent lentivirus, Lenti-X 293 T-cells (Clontech) were transfected with 25 μg of the lentiviral transfer vectors, 25 μg of pCMV-Δ8.9 (packaging plasmid expressing gag and pol), and 5 μg of pMDG-VSVG (vesicular stomatitis virus glycoprotein-pseudotyping envelope) using TransIT Reagent (Mirus Bio LLC), following manufacturer's instructions. One day posttransfection, medium was changed to Ultraculture Serum Free Media (Lonza). The third day posttransfection (or two days after media change), vector supernatants were collected and concentrated by ultrafiltration using Centricon Plus 70 filter units (Millipore) and then filtered through 0.45 μm Spin-X Centrifuge filter tubes (Costar). The vectors were aliquoted and stored at −80°C. The viral titer was calculated by performing serial dilutions of virus on target cells and assaying for fluorescent reporter expression after 3 days.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!