2,296 molecules were screened (Supplementary Table 1 ) and derived from the rolling and curated ICCB-Longwood Screening Facility at Harvard Medical School. All plates used a 384 well format. 704 molecules were chosen for known activity toward PBMCs; these molecules filled up two library plates and originated from the ChemDiv6 library (ChemDiv Inc, San Diego, CA). Another 1592 molecules were chosen from the Selleck bioactive chemical plates (Selleck Chemicals LLC, Houston, TX). The compounds (stored at 10 mM in dessicated conditions) were pinned at a volume of 100 nL, for a final concentration of 33 μM in a 384 well format. 5 μL of 0.3% DMSO(Millipore, Burlington, MA) diluted in DMEM, the negative control, and the TLR9 agonist ODN2395 and the TLR7/8 agonist R848 (both from Invivogen, San Diego, CA), the positive controls, were added to the wells manually for final concentrations of 1μM and 25 μM respectively. PBMCs were then stimulated for 72 hours at 37°C, 5% CO2 in a humidified ThermoScientific Forma CO2 Incubator (Waltham, MA).
Forma co2 incubator
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Forma CO2 incubator is a laboratory equipment designed to provide a controlled environment for cell culture applications. It maintains a stable temperature and carbon dioxide (CO2) concentration to support the growth and proliferation of cells.
Automatically generated - may contain errors
Lab products found in correlation
Embryoscope time lapse incubator, by Vitrolife (2 mentions)
Embryoviewer software, by Vitrolife (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Odn2395, by InvivoGen (1 mentions)
Dulbecco modified eagle medium (dmem), by InvivoGen (1 mentions)
Primovert inverted microscope, by Zeiss (1 mentions)
T75 flask, by Corning (1 mentions)
Phosphate buffered saline (pbs), by Nacalai Tesque (1 mentions)
Tryple select without phenol red, by Thermo Fisher Scientific (1 mentions)
Rabbit complement, by Thermo Fisher Scientific (1 mentions)
Ix83 fv3000, by Olympus (1 mentions)
6 protocols using forma co2 incubator
1
High-Throughput Screening of Immune Modulators
2
hDPSC Expansion and Passaging Protocol
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Cultured medium was removed, and the culture flasks were washed twice with 1 mL of PBS (Nacalai Tesque). The hDPSC products were removed from the flask using 1 mL of a cell removal reagent TrypLE™ select without phenol red (Thermo Fisher Scientific) at 37 °C for 5 min. The removed hDPSC products were collected by centrifugation, as described above, and the cell pellet was diluted in XFM (1 mL; Biological Industries). The hDPSC products (0.25 × 106 per flask) were seeded in a new T-75 flask (Corning) with XFM (10 mL; Biological Industries). When they were 70% confluent, the hDPSC products were passaged again to expand as described above. The medium was exchanged twice a week. The cells were maintained at 37 °C with 5% CO2 in a Forma™ CO2 incubator (Thermo Fisher Scientific) and were inspected daily using a Primovert inverted microscope (Carl Zeiss Microscopy).
3
Sciatic Nerve-Derived Schwann Cell Isolation
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We prepared SCs using the protocol of Weinstein and Wu (2001 (link)). Briefly, 0–3 day-old SD rat sciatic nerves were dissected and minced (Table 2 ), incubated at 37°C in 3 mg/ml collagenase for 30–40 min, and trypsinized at 37°C for 8–10 min. The cells were then cultured at 37°C and 5% CO2 (Thermo Scientific Forma CO2 incubator) in plastic plates coated with poly-L-lysine containing Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum. of the primary SC cultures were treated with cytosine arabinoside at 10 μM. The surviving fibroblasts were then eliminated using polyclonal anti-Thy1.1 antiserum (Sigma; St. Louis, MO, USA) and rabbit complement (Invitrogen; Carlsbad, CA)-mediated lysis. The SC culture obtained using this procedure was >95% pure, as confirmed using S100B and Hochest 33342 immunocytochemistry (ICC).
4
Time-Lapse Embryo Cultivation and Morphokinetic Analysis
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After oocyte retrieval and fertilization, oocytes were cultivated in universal culture medium (Gynemed Medizinprodukte GmbH & Co. KG, Germany) in a Forma CO2 incubator (Thermo Fisher Scientific, USA). After 14–16 h, fertilization check was performed. All normal fertilized embryos with two pronuclei (PN) were then cultured using Embryoslide dishes in Embryoscope® time-lapse incubator (both Vitrolife AB, Sweden) with 21% oxygen concentration. In the PBB conducted group, zygotes were transferred into the Embryoscope after biopsy. With the built-in camera and microscope, images of the developing embryo were taken every 15 min in seven different layers. Definition of morphokinetic parameters was performed according to the criteria proposed by Ciray et al. [8 (link)] (Table 1 ) and was analyzed with software developed for time-lapse image analysis (Embryoviewer® software; Vitrolife AB, Sweden).
Morphokinetic variables and proposed definitions adapted from Ciray et al. (8 (link))
| Time | Definition of expected events |
|---|---|
| t0 | Time of IVF or mid-time of micro/injection (ICSI/IMSI) |
| tPN | Fertilization status is confirmed |
| tPNf | Time of pronuclei disappearance; tPN1f; tPN2f |
| t2 to t9 | 2 to 9 discrete cells |
| tMor | End of compaction process (last frame before cavity formation) |
5
Time-Lapse Embryo Monitoring Protocol
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After oocyte retrieval and fertilization, oocytes were cultured in a Forma CO2 incubator (Thermo Fisher Scientific, Waltham, USA) with universal culture medium (Gynemed Medizinprodukte GmbH & Co.KG, Lensahn, Germany). Fertilization check was performed after a time period of 16–18 hours. Subsequently, embryos with 2 pronuclei (PN) were cultured in an Embryoscope time-lapse incubator (Vitrolife AB, Göteborg, Sweden) at stable conditions of 21% oxygen concentration, 6% CO2 and 37°C. Time-lapse acquisition was set at 15 minute intervals in 9 focal planes. Morphokinetic parameters and parameters of dynamic monitoring of embryo development were described according to the criteria proposed by Ciray and coworkers [23 ] (Table 1 ). Analysis was performed using a software developed for Embryoscope (Embryoviewer software; Vitrolife AB). In addition, irregular events in embryo development such as direct cleavage (a single blastomere divides directly from 1 to 3 cells in less than 5 hours) and reverse cleavage (a blastomere is re-absorbed after cleavage) were annotated according to the description by Rubio et al. [14 (link)].
6
Hypoxia Imaging with Confocal Fluorescence
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An O2 concentration of 1% was controlled by the Thermo Scientific Forma CO2 incubator by means of N2 substitution. Confocal Fluorescence images were obtained on Olympus IX83-FV3000. The fluorescence density was calculated using Image J software (NIH, Bethesda, MD, USA).
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