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> Anatomy > Cell > Null Cell

Null Cell

Null cells are a type of lymphocyte that lack the common lymphocyte surface markers CD3, CD4, and CD8.
They are typically identified by their lack of these markers, which distinguishes them from other lymphocyte subsets.
Null cells play a role in the immune response, though their precise functions are not yet fully understood.
Reseachers can use null cell identification to study immune system dynamics and disorders affecting lymphocyte development and activity.

Most cited protocols related to «Null Cell»

1
Robust Outlier Filtering for scRNA-seq
The Fluidigm assay is sensitive, and owing to the exponential amplification of starting mRNA, even minute contamination can render a measurement unreliable. Similarly, variation in cell preparation can have significant impact on the resulting experiment and data, such as unintentional empty wells, which would distort estimates of . This suggests identifying, and possibly removing outliers before conducting further analysis. We examine both the discrete component and the continuous component in screening for outliers. We define the robust z-transformed positive expression value as

where the median and median absolute deviation (MAD) are calculated, for a given gene, over expressed cells (i.e. ), and is a scaling constant that gives the standard deviation in terms of the MAD for the normal distribution. Next, let be the Bernoulli variance-stabilizing transformation of the proportion of genes expressed in well . Then, we define the robust z-transformed fraction as

where the median, MAD and are as defined previously. This leads to the following steps for filtering:

Remove null cells with no detected genes, i.e. , for all .

Pick threshold for z filtering (); threshold for filtering ().

Calculate and

Remove wells in which genes have OR .

Step 1 removes wells where no cells were loaded, and thus all measured expression values are null. It is important to perform this step first to prevent break-down in the median and MAD estimates for the zeta values in experiments with many amplification or FCM failures. Finally, step 4 removes unreliable wells that either have an extreme proportion of expression or extreme cell gene expression values. The thresholds and control the tolerance to outliers; therefore, typical advice for outlier thresholding applies. Biological replicates, such as the 100-cell replicates described in Section 2.1, permit the assessment of intra-class deviance, and then the thresholds can be selected to minimize this deviance. We present such a calculation in the Supplementary Material. Using this approach, we find that picking works well for the datasets we consider here, see Section 3.
McDavid A., Finak G., Chattopadyay P.K., Dominguez M., Lamoreaux L., Ma S.S., Roederer M, & Gottardo R. (2012). Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments. Bioinformatics, 29(4), 461-467.
Publication 2012
Biological Assay Biopharmaceuticals Cells Gene Expression Genes Immune Tolerance Null Cell RNA, Messenger Strains Transformation, Genetic
2
Controlling Ascertainment Bias in GWAS
Various components of our procedure involve sampling random sets of SNPs from across the genome. While we control for biases in our test statistics introduced by population structure through our matrix, we are also concerned that subtle ascertainment effects of the GWAS process could lead to biased test statistics, even under neutral conditions. We control for this possibility by sampling null SNPs so as to match the joint distribution of certain properties of the ascertained GWAS SNPs. Specifically, we were concerned that the minor allele frequency (MAF) in the ascertainment population, the imputation status of the allele in the HGDP datasets, and the background selection environment experienced at a given locus, as measured by B value [61] (link), might influence the distribution of allele frequencies across populations in ways that we could not predict.
We partitioned SNPs into a three way contingency table, with 25 bins for MAF (i.e. a bin size of 0.02), 2 bins for imputation (either imputed or not), and 10 bins for B value (B values range from 0 to 1, and thus our bin size was 0.1). For each set of null genetic values, we sampled one null SNP from the same cell in the contingency table as each of the GWAS SNPs, and assigned this null SNP the effect size associated with the GWAS SNP it was sampled to match. While we do not assign effect sizes to sampled SNPs used to estimate the covariance matrix (instead simply scaling by a weighted sum of squared effect sizes, which is mathematically equivalent under our assumption that all SNPs have the same covariance matrix), we follow the same sampling procedure to ensure that describes the expected covariance structure of the GWAS SNPs.
For the skin pigmentation GWAS [67] (link) we do not have a good proxy present in the HGDP population, as the Cape Verdeans are an admixed population. Cape Verdeans are admixed with African ancestry, and European ancestry in the sample obtained by [67] (link) (Beleza, pers. comm., April 8, 2013). As such, we estimated genome wide allele frequencies in Cape Verde by taking a weighted mean of the frequencies in the French and Yoruban populations of the HGDP, such that . We then used these estimated frequencies to assign SNPs to frequency bins.
[67] (link) also used an admixture mapping strategy to map the genetic basis of skin pigmentation. However, if they had only mapped these loci in an admixture mapping setting we would have to condition our null model on having strong enough allele frequency differentiation between Africans and Europeans at the functional loci for admixture mapping to have power [126] (link). The fact that [67] (link) mapped these loci in a GWAS framework allows us to simply reproduce the strategy, and we ignore the results of the admixture mapping study (although we note that the loci and effect sizes estimated were similar). This highlights the need for a reasonably well defined ascertainment population for our approach, a point which we comment further on in the Discussion.
Berg J.J, & Coop G. (2014). A Population Genetic Signal of Polygenic Adaptation. PLoS Genetics, 10(8), e1004412.
Publication 2014
African People Alleles Europeans Genome Genome-Wide Association Study Joints Negroid Races Null Cell Reproduction Single Nucleotide Polymorphism Skin Pigmentation
3
Generating Zyxin-Null Fibroblast Cell Lines
Primary mouse embryonic fibroblasts (MEFs) were derived from day 14 embryos of wild-type or zyxin-null mice (backcrossed nine generations into the inbred C57BL/6 line [Charles River Laboratories] to ensure genetic homogeneity, except at the zyxin locus; Hoffman et al., 2003 (link)). Primary MEFs were expanded and frozen as passage 2 stocks. For experiments, the cells were thawed and used within a week (passages 4–6) as early-passage MEFs. To generate immortalized cell lines, cells were cultured from tissue (torso) explants of newborn mice. After spontaneous immortalization (generally passages 8–12), viable cell lines were isolated using cloning cylinders, expanded, and frozen for future use. All cells were grown in high glucose DME supplemented with pyruvate, glutamine, penicillin/streptomycin (Invitrogen), 10% FBS (HyClone) at 37°C, and 5% CO2. Cells were treated with 100 nM of jasplakinolide or DMSO control for 2 h in DME supplemented with 10% FBS (Bubb et al., 2000 (link)). Unless specified in the figure legend, experiments used both primary and immortalized cell lines, with similar results obtained for both. Immortalized cells were used for rescue experiments.
An enhanced GFP-mouse zyxin DNA was inserted into the retroviral vector LINXv-myc (Hoshimaru et al., 1996 (link)) and Phoenix-Eco cells (American Type Culture Collection) were used to generate retrovirus. Zyxin-null fibroblast cell lines were infected and sorted for GFP to select enhanced GFP-zyxin-myc–expressing cells. Caldesmon-6histidine DNA was inserted into pBABEpuro and transiently transfected with Lipofectamine 2000 (Invitrogen).
Hoffman L.M., Jensen C.C., Kloeker S., Wang C.L., Yoshigi M, & Beckerle M.C. (2006). Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodeling. The Journal of Cell Biology, 172(5), 771-782.
Publication 2006
Caldesmon Cell Lines Cells Cloning Vectors Embryo Fibroblasts Freezing Glucose Glutamine Infant, Newborn jasplakinolide lipofectamine 2000 Mice, Knockout Mus Null Cell Penicillins Pyruvate Reproduction Retroviridae Rivers Streptomycin Sulfoxide, Dimethyl Tissues Torso ZYX protein, human
4
Genotyping and Culturing Isogenic Cell Lines
Human LCL cultures GM18870 (G/G, wild-type p53), GM18871 (A/A, homozygous S47), and GM18872 (A/G, heterozygous child from GM18870 and GM18871 parents) were identified for genotype at rs1800371 using the 1000 Genomes Web site and were obtained from the Coriell Institute. These were grown in RPMI supplemented with 15% fetal bovine serum (FBS) and 1% 100 IU/mL penicillin/100 µg/mL streptomycin (pen/strep). The genotype of these lines was confirmed by DNA sequencing. H1299 p53-null human non-small-cell lung carcinoma cells containing a tetracycline regulatory element (H1299 T-Reg) were provided by Steven McMahon (Thomas Jefferson University). The tetracycline-inducible p53 plasmid (Plenti4/TO/V5-DEST) was subjected to site-directed mutagenesis to generate the S47 variant. H1299 T-Reg wild-type or S47 p53 cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM; Cellgro), 1% pen/strep (Cellgro, 30-002-CI), and 10% tetracycline approved FBS (Clontech, 631106). MEFs were obtained from 12.5-d-old Hupki mice and were cultured at 37°C in DMEM with 1% pen/step and 10% FBS (Gemini, 100–106). Etoposide (Sigma, E1383), CDDP (Acros Organics, 193760010), and carboplatin (Sigma, C2538) were used at the indicated concentrations. Doxycycline (BD Biosciences, 631311) was used at a concentration of 100 ng/mL. RSL3 (Aobious, Inc., AO1514), erastin (Sigma Aldrich, 571203-78-6), and Fer-1 (Sigma Aldrich, SML0583) were used at the concentrations indicated.
Jennis M., Kung C.P., Basu S., Budina-Kolomets A., Leu J.I., Khaku S., Scott J.P., Cai K.Q., Campbell M.R., Porter D.K., Wang X., Bell D.A., Li X., Garlick D.S., Liu Q., Hollstein M., George D.L, & Murphy M.E. (2016). An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model. Genes & Development, 30(8), 918-930.
Publication 2016
Carboplatin Cells Child Cisplatin Doxycycline erastin Etoposide Fetal Bovine Serum Genome Genotype Heterozygote Homo sapiens Homozygote Mus Mutagenesis, Site-Directed Non-Small Cell Lung Carcinoma Null Cell Parent Penicillins Plasmids Regulatory Sequences, Nucleic Acid Streptomycin Tetracycline
5
Microscopy for Studying Cell Motility
Cells were placed on a coverslip (18×18 mm, no.1, Matsunami Inc.) and overlaid with a thin agarose sheet as described previously (Yumura et al., 1984 (link)). The coverslip was inverted and placed over a glass slide attached to supports made of double-sided adhesive tape (5×20 mm, 0.3 mm thick). The sample was then sealed with plastic glue (Fastening Systems, USA). Cells were observed under a TIRF microscope of our own composition (Tokunaga et al., 1997 (link); Sako et al., 2000 (link)). An inverted microscope (Olympus IX-70, Japan) was connected to an argon laser (488 nm, H210AL, National Laser Inc.) and a helium–neon laser (543 nm, LHGR-0200, Research Electro Optics Inc.) as previously described (Yumura et al., 2008 (link)). Chromatic aberration was improved by replacing all lenses with achromat lenses. The images were acquired with a cooled CCD camera (Orca ER, Hamamatsu Photonics) and processed with a computer and imaging software (IPlab, Scanalytics). To minimize photobleaching during image acquisition, the intensity of the excitation light was set at minimum. The decrease in the fluorescence intensity after each observation was less than 1–2%, and this level of excitation did not affect cell behavior or motility. Confocal microscopy and photobleaching were performed on a LSM510META microscope (Carl Zeiss) as previously described (Yumura, 2001 (link)). Briefly, the full power of the argon laser (488 nm line) was applied to a particular region of a cell. Changes in fluorescence intensity in the bleached area were monitored with respect to time after background subtraction. The time course of recovery was fitted to the equation for a single exponential rise to maximum using ImageJ software (http://rsbweb.nih.gov/ij), and the half-time recovery was calculated as previously described (Yumura, 2001 (link)).
To observe the movement of beads, cells were placed in a glass-bottomed chamber. A small amount of solution containing carboxylated beads (0.5 µm in diameter, Molecular Probes) was applied to the cells using a micropipette under a phase-contrast inverted microscope (TE300, Nikon). All images were processed and analyzed using ImageJ software.
To detach the cells from the substratum, the external solution was exchanged with an EDTA solution (10 mM NaCl, 10 mM KCl, 2 mM EDTA, 0.1 mM MgCl2, and 2 mM MES, pH 6.3). After 30–60 minutes, all cells were detached from the substratum. To observe the ventral surface of the detached cells and talin A/B null cells by TIRF microscopy, the cells were settled on a glass-bottomed dish and then overlaid with a 2 mm-thick agarose block.
Yumura S., Itoh G., Kikuta Y., Kikuchi T., Kitanishi-Yumura T, & Tsujioka M. (2012). Cell-scale dynamic recycling and cortical flow of the actin–myosin cytoskeleton for rapid cell migration. Biology Open, 2(2), 200-209.
Publication 2012
Argon Ion Lasers Cells Edetic Acid Eye Fluorescence Helium Neon Gas Lasers Hyperostosis, Diffuse Idiopathic Skeletal Lens, Crystalline Light Magnesium Chloride Microscopy Microscopy, Confocal Microscopy, Phase-Contrast Molecular Probes Motility, Cell Movement Null Cell Orcinus orca Sepharose Sodium Chloride Talin

Most recents protocols related to «Null Cell»

1
SAFA-FSH Production and Purification
SAFA-FSH was produced by CHO glutamine synthetase null−/− K1 cell (Horizon Discovery, Waterbeach, UK) and purified using a three step purification protocol—capture for affinity chromatography, intermediate purification for multimodal chromatography, and polishing for cation exchange chromatography, as described previously (14 (link), 15 (link)). After purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC) under native conditions were used to determine the apparent molecular weight and purity.
Kim D., Lee S., Cho Y.H., Kang M.J., Ku C.R., Chi H., Ahn J., Lee K., Han J., Chi S., Song M.Y., Cha S.H, & Lee E.J. (2023). Long-acting recombinant human follicle-stimulating hormone (SAFA-FSH) enhances spermatogenesis. Frontiers in Endocrinology, 14, 1132172.
Publication 2023
Chromatography Chromatography, Affinity Glutamate-Ammonia Ligase High-Performance Liquid Chromatographies Multimodal Imaging Null Cell SDS-PAGE
2
Single-Cell Recording and Marking in Pretectum
We used homemade glass-insulated tungsten microelectrodes with an impedance of 1–3 MΩ for single-cell recording and electrolytic marking of recording sites (Gioanni et al., 1983 (link); Yang et al., 2008b (link)). To record the pretectal neurons in the MIRS light path, we placed the electrode laterally into the left tectum to target the nLM (from the interaural midpoint, A 5.5–6.0 mm, L, 4.0 mm; H, 5.5–6.0 mm). The depth range of recorded neurons was between 2.42 and 4.08  mm. The distance between recording microelectrodes and fiber tips was about 848±351 µm (mean ± SD, n=15, Figure 1E). Extracellular action potentials were filtered using a bandpass filter of 300 Hz to 5 kHz (A-M Systems, Model 1800), and recordings were digitized at 25 kHz (CED, Power 1401 Cambridge electronic design limited). Spike sorting was performed offline (Spike2, Cambridge electronic design limited). Neurons in nLM had high levels of spontaneous activity, and were selective for the direction and velocity of grating motion. Based on this selectivity, we assigned a preferred and null direction to each cell.
At the end of each experiment, recording sites were marked by an electrolytic lesion (positive current of 30–40 µA for 20–30 s), and the animals were given a lethal dose of urethane (4 g/kg). The brain was extracted, postfixed for 24 hr (4% paraformaldehyde with 30% sucrose), immersed in 30% sucrose, and kept at 4 °C for 48 hr before being cut into 50 μm sections through the pretectum (Thermo Scientific Cryotome E). Using a standard light microscope, we reconstructed lesions, fiber tracts, and electrode tracts and verified that all recording sites were confined to the nLM (Figure 1E).
Xiao T., Wu K., Wang P., Ding Y., Yang X., Chang C, & Yang Y. (2023). Sensory input-dependent gain modulation of the optokinetic nystagmus by mid-infrared stimulation in pigeons. eLife, 12, e78729.
Publication 2023
A 300 Action Potentials Animals Brain Cells Electrolysis Fibrosis Genetic Selection Light Light Microscopy Microelectrodes Neurons Null Cell paraform Region, Pretectal Sucrose Tectum Mesencephali Tungsten Urethane
3
Claudin-null EpH4 cell establishment
EpH4 cells were grown in DMEM supplemented with 10% FBS. Claudin-null EpH4 cells were established using the CRISPR-Cpf1 system. The primary and secondary antibodies used for immunofluorescence microscopy and immunoblotting are listed in Supplementary Table 1. Cytochalasin D was added to the cells at a final concentration of 1 µM.
Shigetomi K., Ono Y., Matsuzawa K, & Ikenouchi J. (2023). Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation. Proceedings of the National Academy of Sciences of the United States of America, 120(8), e2217561120.
Publication 2023
Antibodies Cells Claudins Clustered Regularly Interspaced Short Palindromic Repeats Cytochalasin D Immunofluorescence Microscopy Null Cell
4
Proximity Ligation Assay for Cell-Cell Junctions
PLA was performed on EpH4 wild-type and claudin-null cells using the Duolink PLA kit (Sigma-Aldrich). The cells cultured on coverslips were fixed with 100% methanol at −20 °C and then blocked with 1% BSA prepared in PBS for 30 min at RT. Blocked cells were incubated with primary antibodies for 1 h at RT. The combination of primary antibodies was either rabbit anti-JAM-A pAb and mouse β-catenin mAb or rabbit anti–α-catenin and mouse anti-ZO-1. After washing, the samples were incubated with PLA probe solution containing the secondary antibodies for 1 h at 37 °C. Then, the samples were washed and incubated with the ligation solution containing the DNA ligase for 30 min at 37 °C. After ligation, the samples were incubated with the amplification solution containing DNA polymerase and fluorescently labeled nucleotides for 100 min at 37 °C. Finally, the samples were washed, counterstained with DAPI in mounting medium, and observed with a confocal microscope. Images from three fields of view were acquired for each sample, with each field of view containing ≥20 nuclei as identified by DAPI staining. The number of PLA signals was divided by the number of nuclei to calculate the number of PLA signals per cell. Experiments were performed independently three times.
Shigetomi K., Ono Y., Matsuzawa K, & Ikenouchi J. (2023). Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation. Proceedings of the National Academy of Sciences of the United States of America, 120(8), e2217561120.
Publication 2023
Antibodies Catenins Cell Nucleus Cells Claudins CTNNB1 protein, human DAPI DNA-Directed DNA Polymerase DNA Ligases F11R protein, human Ligation Methanol Microscopy, Confocal Mus Nucleotides Null Cell Rabbits Signal Transduction
5
Generation of Claudin-Deficient Cell Lines
pLenti CMV GFP Neo (Addgene Plasmid #17447) was used for the construction of expression vectors. DNA fragments encoding mouse claudin-3, claudin-3 lacking the COOH terminus KDYV (delYV), and claudin-3 carrying C113S, C116S, C192S, and C194S mutations (4S) were subcloned into the pLenti CMV GFP Neo vector. Claudin-null cells were generated by using the CRSPR-Cpf1 system. The target sequences were as follows:
claudin-1 (mouse), 5′-ATCCTGGCTTCTCTGGGATGGAT-3′; claudin-3 (mouse), 5′-GGCCTTCATCGGCAGCAGCATCA-3′; claudin-4 (mouse), 5′-CCTCTTCTGCCCGGAAGCCACCA-3′; claudin-7 (mouse), 5′-GCAGCTCATCATGCCGGTGCTCT-3′; claudin-9 (mouse), 5′-GTGTCCTGTGCCCTGCCACTGTG-3′; and claudin-10b (mouse), 5′-GCCAACCTGTGGAAGATCTGCGT-3′.
Shigetomi K., Ono Y., Matsuzawa K, & Ikenouchi J. (2023). Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation. Proceedings of the National Academy of Sciences of the United States of America, 120(8), e2217561120.
Publication 2023
Claudin-1 Claudin-3 Claudin-4 Claudins Cloning Vectors Mice, Laboratory Mutation Null Cell Plasmids

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1
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
2
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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FBS, or Fetal Bovine Serum, is a commonly used cell culture supplement. It is derived from the blood of bovine fetuses and provides essential growth factors, hormones, and other nutrients to support the growth and proliferation of a wide range of cell types in vitro.
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More about "Null Cell"

Null cells, also known as natural killer (NK) cells, are a type of lymphocyte that lack the common lymphocyte surface markers CD3, CD4, and CD8.
These cells are typically identified by their absence of these markers, which distinguishes them from other lymphocyte subsets.
Null cells play a crucial role in the immune response, though their precise functions are not yet fully understood by researchers.
To study immune system dynamics and disorders affecting lymphocyte development and activity, researchers often utilize null cell identification techniques.
These techniques may involve the use of various cell culture media and reagents, such as Lipofectamine 2000, DMEM, FBS, Lipofectamine 3000, L-glutamine, Penicillin/streptomycin, RPMI 1640 medium, and Penicillin.
Lipofectamine 2000 and Lipofectamine 3000 are transfection reagents commonly used to introduce genetic material into cells, including null cells, to study their functions and interactions within the immune system.
DMEM and RPMI 1640 medium are commonly used cell culture media that provide the necessary nutrients and growth factors for null cells and other lymphocyte subsets.
FBS, or fetal bovine serum, is often added to cell culture media to provide additional growth factors and support the proliferation and differentiation of null cells.
L-glutamine and Penicillin/streptomycin are also common supplements added to cell culture media to support cell growth and prevent bacterial contamination.
By understanding the role of null cells in the immune system and utilizing the appropriate cell culture techniques and reagents, researchers can gain valuable insights into the dynamics of the immune response and develop new strategies for addressing lymphocyte-related disorders.
This knowledge can ultimately lead to advancements in immunology, disease treatment, and overall human health.