For the axonal deformation experiments, the neurons were directly seeded on the PDMS surface. The latter was obtained by spin-coating a bottom glass (Wilco Glass, #1.5) for 1 min at 750 rpm. The coated dish was then plasma-treated for 5 min (Plasma Surface Technology, Diener Electronic), UV-irradiated for 1 hour, and incubated with 500 μl of peanut lectin (1:10 in PBS; Sigma-Aldrich) for 24 hours. Axonal deformation was undertaken by indentation of the substrate with a 1-mm-thick microinjection needle held with a 4D control micromanipulator (uMP-4, Sensapex). The needle tip was brought ≈5 mm away from the axonal tip and pushed vertically to induce substrate indentation. Substrate deformation was determined from the average perpendicular displacement at the middle of the axon. GCaMP activity was measured from the cell body and was normalized to the mKate, Ca2+-independent fluorescence signal (see below). A total of 25 cells were tested, 18 of which showed reproducible responses to substrate indentation.
Peanut lectin
Manufactured by Merck Group
Sourced in United States, Brazil
Peanut lectin is a carbohydrate-binding protein isolated from peanuts. It recognizes and binds to galactose and N-acetylgalactosamine, which are found on the surface of certain cells. Peanut lectin can be used in various laboratory applications, such as cell biology research and glycoprotein analysis.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Chitinase, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Imaging dishes, by MatTek (1 mentions)
L 15 medium, by Merck Group (1 mentions)
L 15 medium, by Thermo Fisher Scientific (1 mentions)
Triazol reagent, by Thermo Fisher Scientific (1 mentions)
Chitinase from streptomyces griseus, by Merck Group (1 mentions)
2 deoxy d glucose, by Merck Group (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
18 mm glass coverslips, by Thermo Fisher Scientific (1 mentions)
Durapore syringe filter, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
10 protocols using peanut lectin
1
Axonal Deformation and Calcium Dynamics
2
Isolation and Culturing of Zebrafish Cone Photoreceptors
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Adult (2 to 6 mo old) zebrafish were dark-adapted overnight and at light onset, were anesthetized in tricaine. Retinas were dissected in a 2:1 mixture of L15 medium (Sigma, St. Louis, MO) and water (hereafter termed L10 medium) and then gently shaken in imaging dishes (MatTek, Ashland, MA) previously coated for 20 min in 0.5 mg/ml peanut lectin (Sigma). Under these conditions, cones break at the myoid and the mitochondria-rich CIS–COS can be cultured for several hours for experimental treatments (Burnside et al., 1993 (link)). CIS–COS were allowed to settle for 1 h before imaging at room temperature. For drug treatment experiments, retinas dissected from an individual fish were separated with one retina undergoing drug treatment and the other retina used as a control.
3
Purification of Metacyclic Leishmania Promastigotes
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The lectin-binding agglutination method described by Saraiva et al. (2005) (link) with some modifications was used to purify metacyclic promastigotes. Briefly, parasite samples from a test culture condition and a control culture condition (the selected medium supplemented with 10% (v/v) FBS, pH 7.0) were collected at the stationary phase of growth by centrifugation at 2,000×g, 20°C for 10 min, washed three times with phosphate-buffered saline (PBS), and resuspended in PBS at 108 cells/mL. These were incubated with 100 μg/mL peanut lectin (Sigma-Aldrich, St Louis, MO, USA) for 30 min at room temperature to purify metacyclic promastigotes. After incubation, cell suspensions were centrifuged at 150×g for 5 min and then the non-agglutinated metacyclic promastigotes were collected from the supernatant. Live metacyclic promastigotes were counted immediately using a haematocytometer and the percentage of metacyclic promastigotes was determined.
4
Leishmania major Friedlin Promastigote Culture
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L. major Friedlin (MHOM/JL/80/Friedlin) were grown as promastigotes in hemoflagellate modified Eagle's medium (HOMEM) supplemented with 10% heat-inactivated FCS at 27°C (9 (link)). Parasite lines deficient in ISP2 and ISP3 (Δisp2/isp3) and the reexpressing line (Δisp2/isp3:ISP2/ISP3) were generated (9 (link)). Purified metacyclic promastigotes were obtained from stationary-phase cultures by washing and incubation with peanut lectin (Sigma-Aldrich, São Paulo, Brazil) at 50 μg/ml for 15 min, followed by centrifugation and recovery in the supernatant (14 (link)).
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5
C. elegans Primary Cell Culture and Imaging
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The protocol for C. elegans primary cell culture was as described previously [20 (link)]. In brief, day 2 adult (D2) worms were lysed to enrich the eggs (lysis solution: 5 mL fresh bleach, 1.25 mL 10 mol/L NaOH, and 18.5 mL sterile H2O). Eggs were digested with 2 mg/mL chitinase (Sigma, catalog no. C6137) and dissociated into single cells. Then the cells were implanted on glass-bottomed cell-culture dishes coated with peanut lectin (Sigma, catalog no. L0881) at a suitable cell concentration. The cells were cultured in L-15 medium (Invitrogen, catalog no. 21083-027) with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen, catalog no. 10082-139), 50 U/mL penicillin, and 50 mg/mL streptomycin (Invitrogen, catalog no. 15140-122). After two days in culture, Ca2+ imaging experiments were performed. The isolated OLL neurons were recognized by expression of Pser-2d::mcherry as well as Pser-2d::GCaMP6s. It has been reported that the posterior ventral dorsal (PVD) neurons sense cold via TRPA-1 channels and detect touch through ENaC channels [21 , 22 ]. Given that PVD and OLL neurons share the same ser-2d promoter, we isolated cells of the trpa-1 mutant strain for temperature-stimulated Ca2+ imaging to exclude PVD neurons. For touch-stimulated Ca2+ imaging in isolated OLL neurons, we immersed the cells in bath solution containing 400 μmol/L amiloride to rule out PVD neurons.
6
Optimized Technique for Intracellular Metabolic Imaging
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2-Deoxy-D-Glucose (Sigma-Aldrich, D8375-5G), Sodium Azide (Sigma Aldrich, S8032-25G), DiD solid (Life Technologie, D7757), Leibovitz’s L-15 medium (Life Technologies, 11415–064), Chitinase from Streptomyces Griseus (Sigma, C6137-25 UN), Fetal Bovine Serum (Invitrogen, 16140–063), Penicillin-Streptomycin (Sigma Aldrich, P4333-100ML), Peanut Lectin (Sigma Aldrich, L0881-10MG), Vectashield mounting medium (Vector Laboratories, H-1000), Triazol reagent (Invitrogen, 15596–026), High capacity RNA-to-cDNA Kit (Applied Biosystem, 43874006), Taqman Universal Master Mix II (Applied Biosystem, 4440043),Taqman probe Ce02485188_m1 (Life Technologies, 4448802), Taqman probe Ce02502871_g1 (Life Technologies, 4448892) Taqman probe Ce02502865_g1 (Life Technologies, 4448892) H2-DCFDA (Sigma-Aldrich, 35845-1G).
7
Isolation and Culture of C. elegans Neurons
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Neurons were isolated from wild type and apm-2 mutants expressing Pglr-1::ha::glr-1::gfp (pzIs12) and cultured as previously described (Christensen et al., 2002 (link); Strange et al., 2007 (link)). Briefly, egg-bearing adult worms were collected from age-synchronized plates and washed with M9 buffer to remove bacteria. Worms were lysed in egg isolation solution (2N NaOH, 20% bleach) for 5 min and washed several times with egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.3). Eggs were harvested by sucrose float and digested under sterile conditions using chitinase (Sigma-Aldrich). Digested eggs were then resuspended in sterile L-15 medium (Invitrogen) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Invitrogen), 50 U/ml penicillin (Invitrogen), and 50 μg/ml streptomycin (Invitrogen). Cells were then gently dissociated from the digested eggs by pipetting the cell suspension against the inside wall of a sterile microcentrifuge tube and were then filtered using a 5-μm Durapore syringe filter (Millipore, Bedford, MA). Cells were seeded onto 18-mm glass coverslips (Thermo Fisher Scientific, Waltham, MA) coated with peanut-lectin (Sigma-Aldrich) and grown in L-15 media with FBS, penicillin, and streptomycin. Cell cultures were maintained in humidified boxes at 20°C, and immunolabeling experiments were conducted 3–4 d postplating.
8
Multicolor Protein Labeling Protocol
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Primary antibodies: 4A12 (rat, ICC: 1:20), 2F8 (rat, WB: 1:50), pcGBA2 (rb, ICC/IHC: 1:2,000) [1 (link),45 (link)], calnexin (Sigma #C4731, WB: 1:20,000), beta-tubulin-CY3 (Sigma #C4585, ICC: 1:200), beta-tubulin (Sigma T4026, WB: 1:1,000), HA (Roche #11867431001, WB: 1:10,000), beta-tubulin III (HISS Diagnostics, MMS-435P, ICC: 1:500, WB: 1:1,000).
Secondary antibodies: WB: IRDye680 and IRDye800 antibodies (LI-COR, 1:20,000); ICC: fluorescently-labeled antibodies (Dianova, 1:500).
Dyes: Alexa Fluor 488 Phalloidin (Molecular Probes, #A12379, ICC: 1:500), MitoTracker (Molecular Probes, M22426, ICC: 0.5 μM), peanut lectin (Sigma, #7381, ICC: 1:100), DAPI (Molecular Probes, D1306, ICC: 1:10,000).
Secondary antibodies: WB: IRDye680 and IRDye800 antibodies (LI-COR, 1:20,000); ICC: fluorescently-labeled antibodies (Dianova, 1:500).
Dyes: Alexa Fluor 488 Phalloidin (Molecular Probes, #A12379, ICC: 1:500), MitoTracker (Molecular Probes, M22426, ICC: 0.5 μM), peanut lectin (Sigma, #7381, ICC: 1:100), DAPI (Molecular Probes, D1306, ICC: 1:10,000).
9
Primary C. elegans Embryo Culture
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Primary cultures of C. elegans embryos were performed according to protocol described previously (Strange et al., 2007 (link)). In brief, worm embryos were isolated from adult hermaphrodites. After removal of the worm eggshells by enzymatic digestion, cells were dissociated and plated onto the dishes precoated with peanut lectin (Sigma). Whole-cell patch clamp was performed 4–6 d after seeding.
10
Measuring Cell Permeability in S. pombe
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Wildtype S. pombe cells were fixed for 15 min or 30 min as above. Fixed and live cells were mounted on coverslips coated with 0.5 mg/mL peanut-lectin (Sigma-Aldrich; L0881-5MG) for for each condition; 1 ROI per cell) and the extracellular environment (6~12 ROIs for each condition) (Fig. S3A). We measured the MSPP of all ROIs including the negative controls (for autofluorescence subtraction), quantified the cell permeability in each condition by calculating (MSPP cytoplasmic -MSPP negative control )/ MSPP extracellular , and reported the mean and standard deviation.
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