The figs and blackthorns were obtained from a local producer in Bragança, Portugal (N 41°47.422, W 6°45.606). The samples were peeled, and the peels of F. carica and the epicarp of P. spinosa were lyophilized (FreeZone 4.5, Labconco), crushed to 20 mesh and stored in a freezer at -20 °C for subsequent analysis. The lyophilized extracts were extracted under the best conditions according to a previous study (Backes et al., 2018; Leichtweis et al., 2019) , relying on an ultrasonic probe apparatus (QSonica sonicators, model CL-334, Newtown, CT, USA), where each plant was extracted with 100 mL of acidified solvent (pH 3, using citric acid). For the fig peel extraction, the solvent used was 100% ethanol (180 g/L, 21 min, 310 W), whilst for blackthorn, the solvent used was a mixture (50:50 v/v) of ethanol:water (75 g/L, 5 min, 400 W). The samples were centrifuged (6000 rpm for 20 min at 10 °C) and filtered through filter paper Whatman n°4 to remove suspended solids. The supernatants were lyophilized (FreeZone 4.5, Labconco) and stored in a refrigerator (-20 °C) for subsequent analyzes.
Freezone 4
Manufactured by Labconco
Sourced in United States, Switzerland, Macao
The FreeZone 4.5 is a lyophilizer, or freeze dryer, designed for laboratory use. It features a 4.5-liter capacity and can operate at temperatures as low as -50°C. The unit is capable of freeze drying a variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
Whatman no 4 paper, by Cytiva (8 mentions)
Whatman no 4 filter paper, by Cytiva (6 mentions)
No 4 filter paper, by Cytiva (4 mentions)
No 4 paper, by Cytiva (4 mentions)
Methacrylic anhydride, by Merck Group (3 mentions)
Rotary evaporator, by Büchi (3 mentions)
Api 3200 qtrap, by Thermo Fisher Scientific (2 mentions)
Jsm 6300 scanning electron microscope, by JEOL (2 mentions)
Mycodev, by Merck Group (2 mentions)
Irg 2959, by Merck Group (2 mentions)
Dibenzocyclooctyne n hydroxysuccinimide ester, by Vector Laboratories (2 mentions)
R 210, by Büchi (2 mentions)
Optima le 80k, by Beckman Coulter (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Qtrap 6500 lc ms ms, by AB Sciex (1 mentions)
Allegra x 12 centrifuge, by Beckman Coulter (1 mentions)
Rotavapor rii, by Büchi (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
205 protocols using freezone 4
1
Extraction and Characterization of Bioactive Compounds from Fig and Blackthorn Peels
2
Achillea millefolium L. Extraction Protocol
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Achillea millefolium L. was collected in Cova de Lua, Bragança, as previously described by Dias et al. (Tozyo et al., 1994) . The sample was lyophilized (FreeZone 4.5, Labconco, Kansas City, MO, USA), reduced to a fine powder (∼20 mesh), homogenized and conserved in deep freezing conditions (-80 °C) for further analysis.
The hydroethanolic extract was obtained by macerating the fine and lyophilized powder (1.5 g) with two subsequent portions of ethanol/ water (80:20, v/v). Each portion was centrifuged at 150 rpm for 1 h at room temperature (RT). The first portion was filtered and the solid residue was extracted again. Both filtered supernatants (Whatman No. 4 filter paper) were then evaporated under vacuum at 35 °C Flawil, Switzerland) . The remaining water residue was lyophilized (FreeZone 4.5 model 7750031, Labconco, KS, USA). The resulting fine powder was homogenized and the crude extract was stored in the dark at RT until tested. Stock solutions of the plant extract were prepared in water and stored at -20 °C until further use.
The hydroethanolic extract was obtained by macerating the fine and lyophilized powder (1.5 g) with two subsequent portions of ethanol/ water (80:20, v/v). Each portion was centrifuged at 150 rpm for 1 h at room temperature (RT). The first portion was filtered and the solid residue was extracted again. Both filtered supernatants (Whatman No. 4 filter paper) were then evaporated under vacuum at 35 °C Flawil, Switzerland) . The remaining water residue was lyophilized (FreeZone 4.5 model 7750031, Labconco, KS, USA). The resulting fine powder was homogenized and the crude extract was stored in the dark at RT until tested. Stock solutions of the plant extract were prepared in water and stored at -20 °C until further use.
3
Extraction and Antimicrobial Evaluation of Graciliflora Leaves
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Leaves of G. Graciliflora Mart. were collected from a rural area of the municipality of Queimadas in the semi-arid region of the state of Paraíba in northeastern Brazil between July and August 2012. The material was cleaned, placed in paper bags and dehydrated in a laboratory incubator (Fanem 330, Fanem Ltda., São Paulo, SP, Brazil) at 40°C. A voucher specimen was deposited in the collection of the Manuel de Arruda Câmara Herbarium of the State University of Paraíba, Campus I, Campina Grande, Brazil (n° 907/ ACAM).
The extraction process involved maceration for five days, using a proportion of 200 grams of dried, ground leaves to one liter of 50% ethanol. The extract was vacuum concentrated in a rotary evaporator Q344M (Quimis Aparelhos Científicos, Diadema, SP, Brazil) at 40°C and the residual portion was freeze dried (Labconco Freezone 4.5, Labconco Corp., Kansas City, MO, USA) [16] (link). were activated in brain heart infusion (BHI) and in sabouraud dextrose agar medium respectively; the plates were incubated at 37ºC (SP Labor Equipamentos Para Laboratórios, São Paulo, SP, Brazil) for 24 h in either an aerobic or microaerophilic atmosphere (Anaerobic Jar Permution, Curitiba, PR, Brazil) [17, 18] .
The extraction process involved maceration for five days, using a proportion of 200 grams of dried, ground leaves to one liter of 50% ethanol. The extract was vacuum concentrated in a rotary evaporator Q344M (Quimis Aparelhos Científicos, Diadema, SP, Brazil) at 40°C and the residual portion was freeze dried (Labconco Freezone 4.5, Labconco Corp., Kansas City, MO, USA) [16] (link). were activated in brain heart infusion (BHI) and in sabouraud dextrose agar medium respectively; the plates were incubated at 37ºC (SP Labor Equipamentos Para Laboratórios, São Paulo, SP, Brazil) for 24 h in either an aerobic or microaerophilic atmosphere (Anaerobic Jar Permution, Curitiba, PR, Brazil) [17, 18] .
4
Lyophilization and Maceration of Leaf and Root Samples
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The previously frozen leaf and root samples were lyophilized to avoid the denaturation of biochemical compounds. This process was carried out in a liophyllizer model Labconco FreeZone 4.5 (Labconco Inc., Kansas City, USA) at a temperature from −45 °C during seven days. Subsequently, the tissue was macerated for further analysis. This lyophilized tissue (LT) was used for all photosynthetic pigments, bioactive compounds, and enzymatic activity analyses.
5
Extraction and Freeze-Drying of A. andensis
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A. andensis ANESC-ST was grown on agar plates (pH 9.0, 1 wt% NaCl for 72 hours at 30°C). Cells were scraped from 10 agar plates with a sterile spatula and washed with 50 mL deionized (DI) water (Millipore). They were subsequently scraped off and dissolved in sterile water for 1 hour. The suspension was centrifuged (10000 g) to remove planktonic bacteria, and the supernatant was filtered through 0.22 μm membrane filters (Pall Corp.). The resultant solution was then freeze-dried (Labconco freezone 4.5, USA).
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A. andensis ANESC-ST was grown on agar plates (pH 9.0, 1 wt% NaCl for 72 hours at 30°C). Cells were scraped from 10 agar plates with a sterile spatula and washed with 50 mL deionized (DI) water (Millipore). They were subsequently scraped off and dissolved in sterile water for 1 hour. The suspension was centrifuged (10000 g) to remove planktonic bacteria, and the supernatant was filtered through 0.22 μm membrane filters (Pall Corp.). The resultant solution was then freeze-dried (Labconco freezone 4.5, USA).
6
Bee Venom Encapsulation in Niosomes
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The bee venom used as the active compound to be encapsulated within the developed niosomes was collected between August and November of 2018 from Apis mellifera intermissa hives located in the northeast region of Morocco. To collect the bee venom, a double-face bee venom collector was used, especially developed for the purpose by the research team. The device was positioned in the hive at one of the outermost, diametrically opposed ends of the beehive, and produced mild electrical impulse shock waves on the beehive, which made the worker bees sting the glass, as a defense mechanism, leaving the bee venom deposited on it. Following the collection process, the venom was meticulously removed from the glass using a scraper and subsequently stored in pharmaceutical-grade vials. Samples were then freeze-dried, in a freeze dryer (Labconco FreeZone 4.5, Labconco Corporation, Kansas City, MO, USA), and kept at −20 °C until further analysis [78 (link)].
7
Harvesting and Drying of Veratrum californicum
8
Bee Venom Collection Protocol
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Fifteen HBV samples were collected from three regions of Morocco, five samples from each region between August and November 2018, Figure 1 b). The specific coordinates of the apiaries can be found in the Supplementary material, Table S2 . Sample NE5 was collected in the same region, but in 2017, and was used to evaluate storage impact. All samples were collected using a double-face bee venom collector, developed in our laboratory with some specific features, and subjected to a patent. The device was placed in the hive at one of the outmost, opposite ends of the beehive. A mild electrical impulse shock was applied (increasing from 0 V to 12 V, then decreases to initial voltage). The beehive’s optimum duration for bee venom collection was set between 30 min to 60 min, early in the morning or at the beginning of the sunset. The venom collection event’s optimum interval in the same colonies was set between 10 to 15 days. After the collection session, the venom was scraped off from the glass with a sharp scraper and conditioned in pharmaceutical-grade vials. The bee venom was then freeze-dried in a Labconco FreeZone 4.5, Labconco corporation (Kansas City, MO, USA), and kept at −20 °C until further analysis.
9
Freeze-drying of Veratrum californicum
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On 3 July 2014, V. californicum plants were gathered from beside the Shindig Trail in the Boise National Forest, Idaho (N 43 45.719″ W 116 05.327″). The above ground plant was discarded, and the roots/rhizomes were put on ice for transportation to the laboratory. A LabConco Freezone 4.5 freeze drying unit (Labconco Corporation, Kansas City, MO, USA) was used to freeze dry the plant parts for 14 h before storage in the freezer in sealed plastic bags [4 (link)].
10
Characterization of Date Varieties from Saudi Arabia and Tunisia
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Five biological replicates of four date varieties at the Tamar stage were collected from different local markets in Saudi Arabia and Tunisia. Ajwa and Anbra dates were obtained from local farms in Medina, Saudi Arabia, and Sukkari dates from Qassim and Hail, Saudi Arabia. Deglet Nour dates were purchased from local farms in Tozeur, Tunisia. These four date varieties have varied origins and post-harvesting conditions. The fruit flesh (F) and seeds (S) of the dates were separately washed from impurities, and lyophilized (Labconco Freezone 4.5) for two days, before finally being ground into fine powder using a freezer/mill grinder.
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