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Reagent a100

Manufactured by ChemoMetec
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Sourced in Denmark, France

Reagent A100 is a laboratory reagent developed by ChemoMetec for use with their cell analysis instruments. It is a core component required for the proper functioning of the instrument and the accurate analysis of cell samples.

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Lab products found in correlation

Reagent b, by ChemoMetec (3 mentions) Nucleocounter nc 200, by ChemoMetec (1 mentions) Nucleocounter, by ChemoMetec (1 mentions) Accutrend plus meter, by Roche (1 mentions) Nucleocounter nc 3000, by ChemoMetec (1 mentions)

4 protocols using reagent a100

1

Bioreactor Cell Growth Monitoring

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Cell growth in the bioreactor was monitored daily for the whole duration of the runs. Three macrocarriers from the top of the fixed-bed were sampled every day through the biomass probe port, resuspended in 1.5 mL of Reagent A100 (Chemometec, Allerod, Denmark), and vortexed for 2 min for cell lysis. Macrocarriers were then discarded and nuclei from the lysed cells were counted using the NucleoCounter NC-200 (Chemometec). Three macrocarriers from the top, middle, and bottom of the fixed-bed were also counted at the end of the culture after the last harvest to assess potential growth differences across the fixed-bed.
Fiol C.R., Collignon M.L., Welsh J, & Rafiq Q.A. (2023). Optimizing and developing a scalable, chemically defined, animal component-free lentiviral vector production process in a fixed-bed bioreactor. Molecular Therapy. Methods & Clinical Development, 30, 221-234.
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2

Quantification of Implant-Adherent and Exudate Cells

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The number of cells adherent to the implant (n = 6–7/group/time point) or in the exudate (n = 8–9/group/time point), that were retrieved from animals, was measured using the NucleoCounter® system (ChemoMetec). To count cells adherent to the implants, retrieved implants were immediately immersed in lysis buffer (100 μL, Reagent A100, ChemoMetec) in a 96-well plate and shaken for 2 min at 500 RPM to detach the cells from the implant surface. Implants were then removed from the lysate before adding the stabilization buffer (100 μL, Reagent B, ChemoMetec). The cell sample was then loaded in a NucleoCassette™ containing propidium iodide that stains nuclei for automatic counting of total cells. To count cells in exudate, an exudate fraction was directly loaded in NucleoCassette™ for dead cell quantification. An additional exudate fraction was diluted 1:1:1 with lysis buffer and stabilization buffer and drawn into NucleoCassette™, as described above, to count total cells.
Ben Amara H., Martinez D.C., Shah F.A., Loo A.J., Emanuelsson L., Norlindh B., Willumeit-Römer R., Plocinski T., Swieszkowski W., Palmquist A., Omar O, & Thomsen P. (2023). Magnesium implant degradation provides immunomodulatory and proangiogenic effects and attenuates peri-implant fibrosis in soft tissues. Bioactive Materials, 26, 353-369.
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3

Cell Fractionation and Extraction

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Cell pellets were resuspended by repeated pipetting in 400 μL Lysis Buffer (Reagent A100, Chemometec, Villeneuve-Loubet, France) before the addition of 400 μL Stabilizing Buffer (Reagent B, Chemometec). After centrifugation at 200 × g for 5 min, 700 μL supernatant was collected as the cytoplasmic fractions and the remaining 100 μL was kept as the nuclear fractions.
François A., Bouzelha M., Lecomte E., Broucque F., Penaud-Budloo M., Adjali O., Moullier P., Blouin V, & Ayuso E. (2018). Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls. Molecular Therapy. Methods & Clinical Development, 10, 223-236.
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4

Microcarrier-based Cell Culture Characterization

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Daily imaging was done by phase contrast microscopy. Cell counts and viability were performed using the Nucleocounter NC3000 (Chemometec) as per manufacturer's instructions for two separate samples. Counts were performed directly on the microcarriers using the reagent A100 and reagent B protocol. Briefly, the cell‐microcarrier suspension was diluted to a 1:1:1 ratio with reagent A100 and reagent B (Chemometec); reagent A100 lyses the cells from the microcarriers releasing the nuclei, while reagent B stabilizes the suspension. The resulting suspension was loaded onto a Nucleocassette Via‐1, preloaded with acridine orange and DAPI and the cassette then transferred Nucleocounter NC3000 machine for processing.
Spent medium samples were collected before and after medium exchanges in the bioreactor and were analyzed for glucose and lactate concentrations on an AccuTrend Plus meter (Roche). Fresh growth medium was used as baseline control.
Based on cell counts, the following parameters were calculated:

Specific growth rate
μ=lnCxtCx0∆t,where μ is the specific growth rate (h−1), Cx(t) and Cx(0) represent cell numbers at the end and start of the culture, t represents time in culture (h).

Doubling time
td=ln2μ,where td is doubling time (h) and μ is the specific growth rate (h−1).

Fold increase
FI=CxtCx0,where Cx(t) represents the maximum cell number and Cx(0) is the initial cell number.

Ochs J., Hanga M.P., Shaw G., Duffy N., Kulik M., Tissin N., Reibert D., Biermann F., Moutsatsou P., Ratnayake S., Nienow A., Koenig N., Schmitt R., Rafiq Q., Hewitt C.J., Barry F, & Murphy J.M. (2022). Needle to needle robot‐assisted manufacture of cell therapy products. Bioengineering & Translational Medicine, 7(3), e10387.
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