Before being filtered through sterile membranes (0.2μm pore size; Millipore, Jinteng, Tianjin, China), we washed litter samples three separate times in a sterile phosphate buffer solution (PBS: NaCl, KCl, Na2HPO4, and KH2PO4). Filtered samples were then sealed in sterile centrifuge tubes before extracting microbial DNA. Following the manufacturer’s instructions, the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, United States) was used to extract microbial DNA in litter samples. Additionally, we used the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States) for DNA quantification. Primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) were used as the internal transcribed spacer (ITS) gene copy numbers for all samples. Sequencing was done at Shanghai Majorbio Bio-pharm Technology (Shanghai, China), using the MiSeq platform (Illumina, Inc., United States). We submitted raw sequencing data to the National Center for Biotechnology Information Sequence Read Archive1 under the project accession number PRJNA764552.
Sterile membrane
Manufactured by Merck Group
Sourced in United States
The Sterile Membrane is a laboratory equipment used for filtration processes. It is designed to remove microorganisms, particulates, and other contaminants from liquids or gases, ensuring a sterile output. The membrane is made of inert materials and is constructed to withstand high-pressure applications.
Automatically generated - may contain errors
Lab products found in correlation
A11037, by Thermo Fisher Scientific (2 mentions)
A11034, by Thermo Fisher Scientific (2 mentions)
A0362, by ABclonal (2 mentions)
A11033, by ABclonal (2 mentions)
E z n a soil dna kit, by Omega Bio-Tek (1 mentions)
Miseq platform, by Illumina (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Alexa flour 488 anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using sterile membrane
1
Microbial DNA Extraction from Plant Litter
2
Seawater Sampling and Processing
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Seawater was collected in various KB areas as previously reported (Pesciaroli et al. 2012) . Briefly, the main sampling sites were an intertidal zone and the nearby sea surface located on the Cape Kindo peninsula in "Velikaja Salma" Bay (Fig. 1). Samples were collected consecutively for 8 days at the minimum tide level from an intertidal pool (samples from P1 to P8) and from the adjacent open seawater surface (samples from M1 to M8). The site location was chosen for its connection with the whole bay water circulation caused by direct exposure to the intense tidal flow. Samples were also collected offshore at different depths and locations (samples from N1 to N4). The physico-chemical characteristics of sample sites are summarized in Table 1. After sampling, 250 mL of water were vacuum-filtered on sterile membranes (0.22 μm, Millipore, USA). Dry membranes were maintained at 4 °C in sterile tubes. Sterile silica gel was added to keep the membrane dry prior to DNA extraction.
3
Photodynamic Therapy with Methylene Blue
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PS methylene blue (Fórmula e Ação, São Paulo, SP, Brazil) was used at the concentration of 0.1 mg/mL for each sample. PS was dissolved in sterile double distilled water and filtered in a sterile membrane (Millipore, São Paulo, Brazil). Source of light used was red laser (Recover, MMOptics®, São Carlos, Brazil), with wavelength of 660 nm, energy density of 26.3 J/cm2, energy of 10 J, potency 100 mW. Periods of 5, 10 and 15 minutes of irradiation were used in an area of 0.56 cm2, that generated an irradiation of 176.9mW/cm2, according to Junqueira et al. protocol (16 (link)).
4
Conjunctival Immune Cell Profiling
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We performed CIC on the subjects' superior tarsal conjunctiva. The CIC samples were collected at the center of the superior tarsal conjunctiva. In preparation for CIC, subjects underwent local anesthesia with 0.4% oxybuprocaine hydrochloride eye drops. Subsequently, a sterile membrane (0.45 μm, Millipore, Boston, MA) was placed on the surface of the palpebral conjunctiva and pressed for a few seconds. To increase the number of collected cells, the palpebral conjunctiva was gently wiped with cotton swabs to keep it dry.
Indirect immunofluorescence was used to observe the staining of CD4+ and CD8+ cells in CIC samples. After fixing the CIC sample with 4% paraformaldehyde, antibodies against CD4 (A0362, 1:200, Abclonal, Wuhan, China) or CD8 (A11033, 1:200, Abclonal) were incubated at 4°C overnight. After washing, we stained CD4 with an Alexa Fluor 488-conjugated anti-rabbit antibody (A-11034, 1:1000, Invitrogen, Shanghai, China) or CD8 with an Alexa Fluor 594-conjugated anti-rabbit antibody (A-11037, 1:1000, Invitrogen) at room temperature for 50 minutes. The cells were observed with a Leica fluorescence microscope (LEICA DMi8, Leica Microsystems, Wetzlar, Germany). Three different fields of each sample were observed and evaluated to count the positively stained cells.
Indirect immunofluorescence was used to observe the staining of CD4+ and CD8+ cells in CIC samples. After fixing the CIC sample with 4% paraformaldehyde, antibodies against CD4 (A0362, 1:200, Abclonal, Wuhan, China) or CD8 (A11033, 1:200, Abclonal) were incubated at 4°C overnight. After washing, we stained CD4 with an Alexa Fluor 488-conjugated anti-rabbit antibody (A-11034, 1:1000, Invitrogen, Shanghai, China) or CD8 with an Alexa Fluor 594-conjugated anti-rabbit antibody (A-11037, 1:1000, Invitrogen) at room temperature for 50 minutes. The cells were observed with a Leica fluorescence microscope (LEICA DMi8, Leica Microsystems, Wetzlar, Germany). Three different fields of each sample were observed and evaluated to count the positively stained cells.
5
Construction of K7 Mutant Strains
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K7(ΔGT-1), K7(ΔGT-2), and K7(ΔwcaJ) were constructed as previously described (Guo et al., 2014 (link)). Briefly, homologous recombination arms of GT-1, GT-2, and wcaJ obtained by overlap extension PCR were amplified and cloned into pUC19 cloning vector. After digestion with Sal I, these sequences used for target gene replacement were individually ligated to pCVD442(Kmr). Followed by electrotransformation into Escherichia coli SM10 λpir, the plasmid bearing strains were mixed with the recipient strain- K. pneumoniae K7 (1:1 v/v). The mixed bacteria were then centrifuged at 6,000 × g for 10 min and suspended with LB medium on a sterile membrane (Millipore, Billerica, MA, United States) at 30°C overnight. The mixed bacteria attached to the membrane were eluted with sterile saline and plated onto LB plates containing 30 μg/ml kanamycin and 100 μg/ml ampicillin. After 12 h of culture, the putative transconjugants of K7(ΔGT-1), K7(ΔGT-2), and K7(ΔwcaJ) were screened and verified by PCR. All primers used in this study are listed in Supplementary Table S1 . The strains and plasmids used in this work are listed in Supplementary Table S2 .
6
Conjunctival Immune Cell Profiling
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After subjects received adequate topical anesthesia with oxybuprocaine hydrochloride eye drops, sterile membrane (0.45 µm; Millipore, Boston, MA, USA) was placed on the surface of the superior tarsal conjunctiva, gently pressed for 5 seconds, and then removed, and stored at −80°C.
Filter membranes were fixed with 4% paraformaldehyde for 1 hour. After washing with phosphate-buffered saline, filters were blocked with 0.1% Triton at room temperature for 20 minutes, and incubated with anti-CD4 antibody (A0362, 1:200; Abclonal, Wuhan, China) or anti-CD8 antibody (A11033, 1:200; Abclonal) at 4°C overnight, followed by incubation with Alexa Flour 488 anti-rabbit secondary antibody (A-11034, 1:1000; Invitrogen, Shanghai, China) for CD4 or Alexa Flour 594 anti-rabbit secondary antibody (A-11037, 1:1000; Invitrogen) for CD8 for 1 hour at room temperature. Stained samples were examined and photographed with a fluorescence microscope (LEICA DMi8; Leica Microsystems, Wetzlar, Germany) at 400 times magnification. Three fields of view for each sample were randomly chosen, and images were analyzed manually using Image J software to count DAPI+ cells (blue, total cell number), CD4+ cells (green), and CD8+ cells (red). Then, the percentage of CD4+ DAPI+ cells and CD8+ DAPI+ cells was calculated for subsequent statistical analysis.
Filter membranes were fixed with 4% paraformaldehyde for 1 hour. After washing with phosphate-buffered saline, filters were blocked with 0.1% Triton at room temperature for 20 minutes, and incubated with anti-CD4 antibody (A0362, 1:200; Abclonal, Wuhan, China) or anti-CD8 antibody (A11033, 1:200; Abclonal) at 4°C overnight, followed by incubation with Alexa Flour 488 anti-rabbit secondary antibody (A-11034, 1:1000; Invitrogen, Shanghai, China) for CD4 or Alexa Flour 594 anti-rabbit secondary antibody (A-11037, 1:1000; Invitrogen) for CD8 for 1 hour at room temperature. Stained samples were examined and photographed with a fluorescence microscope (LEICA DMi8; Leica Microsystems, Wetzlar, Germany) at 400 times magnification. Three fields of view for each sample were randomly chosen, and images were analyzed manually using Image J software to count DAPI+ cells (blue, total cell number), CD4+ cells (green), and CD8+ cells (red). Then, the percentage of CD4+ DAPI+ cells and CD8+ DAPI+ cells was calculated for subsequent statistical analysis.
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