Following resection, a random piece was selected from viable tumor tissue, minced, and viably frozen. On the day of the sorting, the sample was thawed and dissociated into a single-cell suspension in AdDF + ++ (Advanced DMEM/F12 containing 1× Glutamax, 10 mM HEPES and antibiotics) containing Collagenase 1a (1 mg/mL, Sigma, C9407) and DNase (0.25 µg/mL, Stemcell), supplemented with Rho-kinase inhibitor Y-27632 (10 µM, Abmole). The samples were digested on an orbital shaker for 30 min at 37 °C. The suspension was washed first with AdDF + ++ and next with MACS buffer (PBS pH 7.2 + 2 mM EDTA + 0.5% Bovine Serum Albumine), followed each time by centrifugation at 300 × g. Viable single cells were sorted based on forward/side scatter properties and DAPI/DRAQ5 staining using FACS (MoFlo Astrios EQ, Beckman Coulter) into 384-well plates (Biorad) containing 10 µl mineral oil (Sigma) and 50 nl of RT primers.
384 well plate
Manufactured by Bio-Rad
Sourced in United States, Italy
The 384-well plate is a laboratory equipment used for high-throughput screening and sample analysis. It is a flat-bottomed plate with 384 individual wells, designed to hold small volumes of liquid samples for various applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Iq sybr green supermix, by Bio-Rad (5 mentions)
Biorad cfx 384 real time system c1000 thermal cycler, by Bio-Rad (4 mentions)
Quantitect reverse transcription kit, by Qiagen (4 mentions)
Mineral oil, by Merck Group (3 mentions)
Fc block cd16 32, by Cytek Biosciences (3 mentions)
Le sh800 cell sorter, by Sony (3 mentions)
Sytox blue, by Thermo Fisher Scientific (3 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (3 mentions)
Rneasy, by Qiagen (3 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (3 mentions)
Cfx384 real time system, by Bio-Rad (3 mentions)
Nextseq 500 sequencer, by Illumina (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Y 27632, by AbMole (1 mentions)
Moflo astrios eq, by Beckman Coulter (1 mentions)
Dnase, by STEMCELL (1 mentions)
Collagenase 1a, by Merck Group (1 mentions)
Dna away, by Thermo Fisher Scientific (1 mentions)
Facsmelody, by BD (1 mentions)
27 protocols using 384 well plate
1
Single-Cell Isolation from Tumor Tissue
2
Single-cell sorting of E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli K12 MG1655 (DSMZ 18039) was cultured in 1 mL of Luria Bertani (LB) broth at 30 °C and 750 rpm with the Thermomixer Comfort (Eppendorf, Hamburg, Germany) to the exponential growth phase (~4 h; OD600 of ~2.2–2.6). From this point forward, cells were processed in a UV-decontaminated ISO 4 cleanroom. Equipment and gloves were decontaminated with DNA AWAY (Thermo Fisher Scientific, Waltham, MA, USA). Consumables were UV treated for 1 hr in a Crosslinker and 1 × PBS was UV treated for 6 h in a 254 nm shortwave ultraviolet crosslinker at 0.12 Joules/I2 (Analytik Jena GmbH, Jena, Germany).
A BD FACSMelody (Becton-Dickson, Franklin Lakes, NJ, USA), fitted with a 100 µM nozzle and equipped with a 488 nm laser for excitation was used to sort single cells. Cells were first diluted to approximately 106 cells mL−1 with sterile 1X PBS to ensure an event rate of <1000 events/s. Gates were defined on side-scatter (cell complexity) and forward-scatter (cell-size). Cells were sorted in single-cell mode into 384-well plates (Bio-Rad, Hercules, CA, USA) containing no sorting buffer (i.e., dry sorting). Plates were sealed with Microseal B (Bio-Rad, Hercules, CA, USA) and stored at −80 °C.
A BD FACSMelody (Becton-Dickson, Franklin Lakes, NJ, USA), fitted with a 100 µM nozzle and equipped with a 488 nm laser for excitation was used to sort single cells. Cells were first diluted to approximately 106 cells mL−1 with sterile 1X PBS to ensure an event rate of <1000 events/s. Gates were defined on side-scatter (cell complexity) and forward-scatter (cell-size). Cells were sorted in single-cell mode into 384-well plates (Bio-Rad, Hercules, CA, USA) containing no sorting buffer (i.e., dry sorting). Plates were sealed with Microseal B (Bio-Rad, Hercules, CA, USA) and stored at −80 °C.
3
Single-cell RNA-seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared according to the SORT‐seq method.28 Viable single cells were sorted based on forward/side scatter properties and DAPI staining using FACS (into 384‐well plates [Biorad] containing 10 μl mineral oil [Sigma]) and 50 nl of RT primers. Samples were subsequently processed into Illumina sequencing libraries as described.28 Libraries were sequenced paired end at 75 bp read length using the Illumina NextSeq500 sequencer.
4
Adrenal Gland Single-Cell RNA-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were collected at time points E13.5, E14.5, E17.5, E18.5, P1, and P5. Adrenal glands were isolated and dissociated into single cells. Adrenal glands were collected in PBS containing 1 mg/mL collagenase/dispase (Roche) and sheared using a 1-mL pipette on ice until single-cell suspension was observed. The enzymatic digestion was stopped by the addition of basic medium (Advanced DMEM/F12 [Gibco] containing N2 [1×; Gibco], B27 [1×; Gibco], Hepes [10 mM; Gibco], penicillin–streptomycin [100 U/mL; Gibco], Glutamax [1×; Gibco], N-acetyl cysteine [1.25 mM; Sigma]), and the suspension was centrifuged at 250 × g before being resuspended in PBS. All centrifugation steps were done at 4 °C, with the samples kept on ice for the duration of the experiment.
Single-cell suspensions were stained using 1 µM DAPI (Sigma). Viable single cells were sorted based on forward/side-scatter properties and DAPI staining (gating strategy shown inSI Appendix, Fig. S7 ) using FACS (FACSJazz; BD Biosciences) into 384-well plates (Bio-Rad) containing 10 µL of mineral oil (Sigma) and 50 nL of RT primers. Libraries were prepared using the Sort-seq method (18 (link)) and subjected to paired-end sequencing with 75-bp read length using the Illumina NextSeq500 sequencer.
Single-cell suspensions were stained using 1 µM DAPI (Sigma). Viable single cells were sorted based on forward/side-scatter properties and DAPI staining (gating strategy shown in
5
CRISPR-Cas9 Targeting of Mouse Dnmt1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Six gRNA sequences targeting three exons of mouse Dnmt1 were used as described previously42 (link). Phosphorylated BbsI compatible restriction overhangs were added to gRNA top and bottom oligos and resuspended at 100 μM in nuclease-free water. Annealing of the oligos was performed in 1× ligation buffer (NEB) using the following program: 97 °C for 5 min, ramp down by 1 °C per 1 min to 20 °C. The pX330 CRISPR-Cas9-GFP gRNA plasmid was a kind gift from Eva van Rooij and mixed with 0.1 μM gRNA oligo. The reaction was simultaneously digested with BbsI (NEB) and ligated with T4 DNA ligase (NEB) overnight at 16 °C. Ligation reactions were transformed into DH5α competent cells and subsequently sequenced using Sanger dideoxy sequencing to confirm the correct insert. All six pX300-gRNA plasmids were pooled and 1 μg was transfected into 2 million E14tg2a cells using Lipofectamine (Life Technologies). A separate pX300 empty vector was also transfected into E14tg2a to serve as a negative control. Two days later, single GFP-positive cells were sorted into 384-well plates (BioRad) and subjected to scMspJI-seq.
6
Quantitative Real-Time PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 20-μL reaction of 5-μL cDNA and 15-μL IQ Multiplex Powermix containing the primers and probes for each sample was run in triplicate on a 384-well plate (see Table 1 for concentrations). Positive control stocks from cell culture, 9 serial dilution (1:10) standards (IDT DNA), and 2 nontemplate controls were included for each run. The CFX384 Real-Time System thermocycler was used under the following conditions: 52°C for 2 minutes, and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The baseline threshold of 300 relative fluorescence units was used to determine the Ct for each reaction. A Ct <37 was regarded as positive and samples with ≥2 of 3 reactions below the threshold of 37 were considered positive and set aside for further analysis. The Multiplex Powermix, 384-well plates, and thermocycler were sourced from Bio-Rad.
7
Single-cell sorting of lung cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung cells were plated at 1 × 106 cells per well and stained with Fc block (CD16/32, 1:100, Tonbo Biosciences) for 30 min on ice. Cells were surface stained with the endothelial marker CD31 (1:100, clone: MEC3.1, eBiosciences), epithelial marker CD326 (1:100, clone: CD326, eBiosciences), and immune marker CD45 (1:100, clone: F11, eBiosciences) for 30 min on ice. The live/dead dye, SYTOX Blue (ThermoFisher), was added to cells and incubated for 3 min prior to sorting into 384-well plates (Bio-Rad Laboratories, Inc) prefilled with lysis buffer using the Sony LE-SH800 cell sorter (Sony Biotechnology Inc), a 100μm sorting chip (Catalog number: LE-C3110) and ultra-purity mode. Single color controls were used to perform fluorescence compensation and generate sorting gates. 384-well plates containing single cells were spun down, immediately placed on dry ice and stored at -80C.
8
Quantitative RT-PCR Analysis of PtovoA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 µl of a 1 : 2 dilution of cDNA was used as template to amplify the PtovoA transcript using 0.4 µM final concentration of the primers ovo1fw and ovo1rv, using RPS (Ribosomal protein small subunit 30S; ID 10847) as reference gene [49 (link)]. Real-Time PCR amplification was performed using Power SYBR Green PCR Master Mix 2X (Applied Biosystem) in a final volume of 10 µl. Each reaction was tripled for both genes in each sample using 384- well plates (BioRad) in the ViiA 7 Real-Time PCR System (Thermo Fischer).
Data obtained were analysed with the ViiA 7 Real-Time PCR System software, and fold-changes were obtained with the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) [50 (link)].
Data obtained were analysed with the ViiA 7 Real-Time PCR System software, and fold-changes were obtained with the Relative Expression Software Tool-Multiple Condition Solver (REST-MCS) [50 (link)].
9
Single-cell sorting of lung cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lungs were plated at 1 × 106 cells per well and stained with Fc block (CD16/32, 1:100, Tonbo Biosciences) for 30 min on ice. Cells were surface stained with the endothelial marker CD31 (1:100, eBioscience/ThermoFisher), epithelial marker Epcam (1:100, eBioscience/ThermoFisher), and immune marker CD45 (1:100, eBioscience/ThermoFisher) for 30 min on ice. The live/dead dye, Sytox Blue (Invitrogen), was added to cells and incubated for 3 min prior to sorting into 384-well plates (Bio-Rad Laboratories, Inc) prefilled with lysis buffer using the Sony LE-SH800 cell sorter (Sony Biotechnology Inc), a 100 μm sorting chip (Catalog number: LE-C3110) and ultra-purity mode. Single color controls were used to perform fluorescence compensation and generate sorting gates. 384-well plates containing single cells were spun down, immediately placed on dry ice and stored at − 80 ºC.
10
SARS-CoV-2 Viral Load Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral loads in the respiratory tract, evaluated as a copies of subgenomic (sg)E and sgN RNA in the sample, were measured as previously reported27 (link),53 (link). BAL fluid and nasal swabs were stored in RNAzol BD (Molecular Research Center) and PBS until use, respectively. Total RNA was extracted with the RNAzol BD column kit (Molecular Research Center). TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems), gene-specific primers (sgLeadSARSCoV2_F: 5′-CGATCTCTTGTAGATCTGTTCTC-3′, E_Sarbeco_R: 5′-ATATTGCAGCAGTACGCACACA-3′, wtN_R: 5′-GGTGAACCAAGACGCAGTAT-3′) and probes (E_Sarbeco_P: 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′, wtN_P: 5′-FAM-TAACCAGAATGGAGAACGCAGTGGG-BHQ1-3′) were used for the RT-qPCR reaction in 384-well plates (Bio-Rad). The total volume of the reaction was 20 μL, and the sample volume was 3.33 μL. CFX384 Touch Real-Time PCR Detection System (Bio-Rad) was used for amplifications. The lower limit of quantification was 50 copies/reaction.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!