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Macs dead cell removal kit

Manufactured by Miltenyi Biotec
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Sourced in Germany

The MACS Dead Cell Removal Kit is a laboratory equipment product designed to efficiently remove dead cells from cell samples. It utilizes magnetic beads coated with antibodies that specifically bind to dead cells, allowing for their separation and removal from the sample using a magnetic field. The core function of this kit is to provide a reliable and effective method for isolating viable cells from cell preparations.

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Lab products found in correlation

Calcein am, by Thermo Fisher Scientific (8 mentions) Macs tissue storage solution, by Miltenyi Biotec (8 mentions) Red blood cell lysis buffer, by Miltenyi Biotec (8 mentions) Draq7, by BD (7 mentions) Dnase 1, by Worthington (6 mentions) 35 μm cell strainer, by BD (4 mentions) Red blood cell lysis buffer, by Solarbio (3 mentions) Collagenase 4, by Merck Group (2 mentions) Trypsin inhibitor, by Merck Group (2 mentions) 70 μm cell strainer, by BD (2 mentions) Collagenase 4, by Worthington (2 mentions) Collagenase 1, by Worthington (2 mentions) Collagenase type 1, by Merck Group (2 mentions) Collagenase type 2, by Merck Group (2 mentions) Collagenase type 4, by Merck Group (2 mentions) Octo dissociator, by Miltenyi Biotec (2 mentions) Tumor dissociation kit, by Miltenyi Biotec (2 mentions) Ack lysis buffer, by Quality Biological (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)

40 protocols using macs dead cell removal kit

1

FACS Sorting and scRNA-seq of Immune Cells

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On the day of FACS sorting, cells were rapidly thawed at 37 °C and transferred to complete RPMI media. Live-cell enrichment was performed using MACS Dead Cell Removal Kit (Miltenyi Biotec) following the manufacturer’s instructions. Red blood cells were further depleted by negative selection using CD235a Microbeads (Miltenyi Biotec) and MACS LS columns (Miltenyi Biotec), following the manufacturer’s instructions.
For FACS sorting, cells were stained with Zombie Aqua to exclude dead cells and the cocktail of antibodies for 30 min at 4 °C. Cells were centrifuged for 5 min at 300 × g, 4 °C, resuspended in 500 μl of 5% FBS in PBS and subsequently filtered into polypropylene FACS tubes.
Immune cells were sorted as live, CD45 + ; MDSC were sorted as live, CD45 + , Lineage- (Lin: CD3, CD56, CD19), CD33 + , HLA-DR-/low (Supplementary Data 22 and Supplementary Fig. 1A). Cells were sorted into a 1.5-ml tube, counted and submitted for 10x scRNA-seq library preparation.
De Zuani M., Xue H., Park J.S., Dentro S.C., Seferbekova Z., Tessier J., Curras-Alonso S., Hadjipanayis A., Athanasiadis E.I., Gerstung M., Bayraktar O, & Cvejic A. (2024). Single-cell and spatial transcriptomics analysis of non-small cell lung cancer. Nature Communications, 15, 4388.
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2

Tissue Dissociation and Single-Cell Isolation

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Tissue was surgically minced on a laboratory sterile table, and tissue fragments were preserved in MACS tissue storage until processing.
The tissue samples were processed as described below. Briefly, samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1 mm3) on ice, and enzymatically digested with 500 U/ml collagenase I (SangonBiotech), 150 U/ml collagenase II (SangonBiotech), 50 U/ml collagenase IV (SangonBiotech), 0.1 mg/ml hyaluronidase (SangonBiotech), 30 U/ml DNaseI (SangonBiotech), and 5% Fetal Bovine Serum Origin South America (Yeasen) for 60 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300 g for 5 min. After washing with PBS containing 0.04% BSA, the cell pellets were re-suspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained for viability assessment using Calcein-AM (Thermo Fisher Scientific) and Draq7 (BD Biosciences). The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).
Li Z., Ye D., Dai L., Xu Y., Wu H., Luo W., Liu Y., Yao X., Wang P., Miao H., Xu J, & Liang W. (2022). Single-Cell RNA Sequencing Reveals the Difference in Human Normal and Degenerative Nucleus Pulposus Tissue Profiles and Cellular Interactions. Frontiers in Cell and Developmental Biology, 10, 910626.
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3

Single-cell RNA-seq from Tissue Samples

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scRNA-seq experiments were performed by experimental personnel in the laboratory of Novel Bio Co, Ltd. The tissues were surgically removed and kept in MACS Tissue Storage Solution (Miltenyi Biotec) until processing. Tissue samples were processed as described below. Briefly, samples were first washed with PBS, minced into small pieces (∼1 mm3) on ice, and enzymatically digested with 125 U/mL collagenase IV (Sigma), 25 U/mL collagenase I (Sigma), and 25 U/mL DNase I (Worthington) for 30 minutes at 37°C, with agitation. After digestion, samples were sieved through a 40-μm cell strainer, and then centrifuged at 300 × g for 5 minutes. The supernatant was removed and the pelleted cells were suspended in red blood cell lysis buffer (Solarbio) to lyse the red blood cells. The cells were resuspended in RPMI1640 medium containing 10% FBS and refiltered through a 35-μm cell strainer. Dissociated single cells were then stained with acridine orange/propidium iodide for viability assessment using a Countstar Fluorescence Cell Analyzer. The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).
Xu J., Fang Y., Chen K., Li S., Tang S., Ren Y., Cen Y., Fei W., Zhang B., Shen Y, & Lu W. (2022). Single-Cell RNA Sequencing Reveals the Tissue Architecture in Human High-Grade Serous Ovarian Cancer. Clinical Cancer Research, 28(16), 3590-3602.
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4

Single-cell RNA-seq of Orbital Connective Tissues

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Single-cell suspensions from orbital connective tissues were derived from 1 TAO patient and 1 healthy control as previously described (30 (link)). Single cells were isolated and lysed with collagenase buffer composed of 5 mg/mL collagenase IV and 10 U/mL DNase I at 37°C for 50 minutes (all from Sigma-Aldrich, St. Louis, USA). Subsequently, filtration and red blood cell removal were performed using the MACS Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA was then reverse-transcribed into cDNA libraries using a 10x Genomics Chromium machine following the manufacturer’s instructions. DNA sequencing was performed on a NovaSeq 6000 Sequencing System (Illumina, CA, USA) using a paired-end 150 sequencing mode. The Illumina output was processed using Cell Ranger 3.0.2 (10×Genomics, CA, USA). Cells of sufficient complexity were clustered and t-distributed stochastic neighbor embedding (t-SNE) plots were generated for visualization using the Seurat R package (31 (link)).
Wu P., Lin B., Huang S., Meng J., Zhang F., Zhou M., Hei X., Ke Y., Yang H, & Huang D. (2022). IL-11 Is Elevated and Drives the Profibrotic Phenotype Transition of Orbital Fibroblasts in Thyroid-Associated Ophthalmopathy. Frontiers in Endocrinology, 13, 846106.
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5

Dissociation and Purification of Cells

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Day 9 cultures from both protocol1 (RA) and protocol3 (RA + BMP4) were dissociated with 0.25% cold trypsin for 5 min. Trypsin activity was stopped with the addition of trypsin inhibitor (Sigma-Aldrich) in 1:1 ratio. Cells were then pelleted by centrifugation at 1000RPM and resuspended in 1x PBS. The ratio of viable cells was determined using Trypan blue staining. Dead cells were removed using MACS dead cell removal kit (Miltenyi Biotec Inc.) by following the manufacturer’s protocol. Eluted live cells were then suspended in 1X PBS containing 0.04% BSA solution (400μg/ml) for the library preparation.
Gupta S., Kawaguchi R., Heinrichs E., Gallardo S., Castellanos S., Mandric I., Novitch B.G, & Butler S.J. (2022). In vitro atlas of dorsal spinal interneurons reveals Wnt signaling as a critical regulator of progenitor expansion. Cell reports, 40(3), 111119.
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6

Dissociation and Purification of Cells

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Day 9 cultures from both protocol1 (RA) and protocol3 (RA + BMP4) were dissociated with 0.25% cold trypsin for 5 min. Trypsin activity was stopped with the addition of trypsin inhibitor (Sigma-Aldrich) in 1:1 ratio. Cells were then pelleted by centrifugation at 1000RPM and resuspended in 1x PBS. The ratio of viable cells was determined using Trypan blue staining. Dead cells were removed using MACS dead cell removal kit (Miltenyi Biotec Inc.) by following the manufacturer’s protocol. Eluted live cells were then suspended in 1X PBS containing 0.04% BSA solution (400μg/ml) for the library preparation.
Gupta S., Kawaguchi R., Heinrichs E., Gallardo S., Castellanos S., Mandric I., Novitch B.G, & Butler S.J. (2022). In vitro atlas of dorsal spinal interneurons reveals Wnt signaling as a critical regulator of progenitor expansion. Cell reports, 40(3), 111119.
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7

Enumerating Hematopoietic Colonies

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Supernatants of hematopoietic differentiation cultures were harvested on day 12, filtered through a 70 µm strainer, and live cells were purified using the MACS dead cell removal kit (Miltenyi). After Trypanblue exclusion cell counting in a Bürker-Türk chamber, 2000 cells were seeded per well of a 6-well plate into 300 µL Iscove’s Modified Dulbecco’s Medium (IMDM) containing 2% fetal bovine serum (both from Thermo Scientific) and 3 mL MethoCult semisolid medium either containing an enriched cytokine cocktail or none at all (STEMCELL Technologies H4435 or H4230, respectively). Colonies were enumerated 12 to 14 days later in a 3D microscope under dark field illumination. Live cell yields per well of 12-well plates were compared using unpaired one-way ANOVA with Tukey post hoc test assuming Gaussian distribution and equality of variances (GraphPad Prism 8). Colony pictures were acquired with an EVOS XL core microscope and 4× phase contrast objective (Thermo Scientific).
Fortschegger K., Husa A.M., Schinnerl D., Nebral K, & Strehl S. (2021). Expression of RUNX1-JAK2 in Human Induced Pluripotent Stem Cell-Derived Hematopoietic Cells Activates the JAK-STAT and MYC Pathways. International Journal of Molecular Sciences, 22(14), 7576.
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8

CD56+ Cell Depletion and Kinetics

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MACS Dead cell removal kit and CD56 MicroBeads (Miltenyi Biotec) were used for CD56-positive cell depletion on day 0 CAR T cell products. The CD56-depleted CAR T cell product was subjected to CAE protocol as described above and the frequency of CD56+ T cells was assessed by flow cytometry.
The out-competition model assumes that initial depletion of the NK-like-T cell population would result in altered kinetics of NK-like-T cell abundance over time compared to a non-depleted control group, whereas transitioning assumes similar kinetics between the control and depleted groups. As shown in Figure S6F, in case of out-competition by the CD56-positive cell subset (left panel), the frequency of CD56 in the CD56-depleted cultures increase at a lower rate than in the controls. This growth can be expressed by the formula PT = (P0 − d) × kt. On the other hand, if T cell are transitioning into NK-like-T cells, (S6F, right panel), the frequency of CD56 in the cocultures would increase at the same rate over the time, independently of the initial depletion of the CD56 at the start of the coculture, which can be expressed as PT = (P0 − d) + k × t. PT: percentage CD56-positive cells at time “T”. P0: Percentage CD56-positive cells at time zero. t: time of in vitro stimulation [Days]. k: transition constant. D: percentage CD56-positive cells depleted.
Good C.R., Aznar M.A., Kuramitsu S., Samareh P., Agarwal S., Donahue G., Ishiyama K., Wellhausen N., Rennels A.K., Ma Y., Tian L., Guedan S., Alexander K.A., Zhang Z., Rommel P.C., Singh N., Glastad K.M., Richardson M.W., Watanabe K., Tanyi J.L., O’Hara M.H., Ruella M., Lacey S.F., Moon E.K., Schuster S.J., Albelda S.M., Lanier L.L., Young R.M., Berger S.L, & June C.H. (2021). An NK-like CAR T cell transition in CAR T cell dysfunction. Cell, 184(25), 6081-6100.e26.
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9

Tissue Dissociation and Single-Cell Isolation

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The tissue samples were processed as described below.57 (link) Briefly, samples were first washed with PBS, minced into small pieces (approximately 1 mm3) on ice and enzymatically digested with 1 mg/mL collagenase II (Worthington) for 30 min at 37°C, with agitation. After digestion, samples were sieved through a 70 μm cell strainer, and centrifuged at 300g for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS containing 0.04% BSA, the cell pellets were resuspended in PBS containing 0.04% BSA and re-filtered through a 35 μm cell strainer. Dissociated single cells were then stained with AO/PI for viability assessment using Countstar Fluorescence Cell Analyzer. The single-cell suspension was further enriched with a MACS dead cell removal kit (Miltenyi Biotec).
Liao J., Wang J., Xu Y., Wu Y., Wang M., Zhao Q., Tan X., Meng Y., Wei L, & Huang A. (2023). LAPTM4B-YAP loop feedback amplification enhances the stemness of hepatocellular carcinoma. iScience, 26(6), 106754.
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10

Single-cell RNA-seq of p53 WT and KO cells

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All libraries generated using 10x scRNAseq are listed in Supplementary Table 8. Suspensions of 184-hTERT p53wt and KO cells were fixed with 100% ice-cold methanol prior to preparation for scRNAseq. Single cell suspensions were loaded onto the 10x Genomics single cell controller and libraries prepared according to the Chromium Single Cell 3’ Reagent Chemistry kit standard protocol. Libraries were then sequenced on an Illumina Nextseq500/550 with 42bp paired-end reads, or a HiSeq2500 v4 with 125bp paired-end reads. 10x Genomics CellRanger, V3.0.2 (V3 chemistry), was used to perform demultiplexing, alignment and counting.
Viable frozen tumour clumps and fragments were incubated with digestion enzymes as with DLP+ single cells preparation (as above) and the cells were resuspended in 0.04% BSA in PBS. Dead cells were removed using the Miltenyi MACS Dead Cell Removal kit and cells were processed as previously described37 (link). To avoid processing artifacts and dissociation methods, the timings were tightly controlled between the samples. Library construction of the samples at the same time point was performed on the same chips. Library construction sample batch groupings are listed in Supplementary Table 7.
Salehi S., Kabeer F., Ceglia N., Andronescu M., Williams M.J., Campbell K.R., Masud T., Wang B., Biele J., Brimhall J., Gee D., Lee H., Ting J., Zhang A.W., Tran H., O’Flanagan C., Dorri F., Rusk N., de Algara T.R., Lee S.R., Cheng B.Y., Eirew P., Kono T., Pham J., Grewal D., Lai D., Moore R., Mungall A.J., Marra M.A., McPherson A., Bouchard-Côté A., Aparicio S, & Shah S.P. (2021). Clonal fitness inferred from timeseries modeling of single cell cancer genomes. Nature, 595(7868), 585-590.
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