The largest database of trusted experimental protocols

Live dead fixable red

Manufactured by Thermo Fisher Scientific
Sourced in United States

The LIVE/DEAD Fixable Red Dead Cell Stain Kit is a fluorescent dye-based reagent designed to identify dead cells in a sample. The dye reacts with free amines in the cell, producing a red fluorescent signal that can be detected using flow cytometry or fluorescence microscopy. The kit allows for the reliable identification of dead cells in a variety of applications, including cell viability analyses and cell-based assays.

Automatically generated - may contain errors

5 protocols using live dead fixable red

1

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
For the human cell lines, FACS analysis was performed as follows: Cells were scraped upon reaching up to 90% confluence. Cell pellets were then washed and incubated with HLA antibodies at a 1:10 ratio in FACS buffer containing 1% FBS, 0.1% sodium azide in PBS. Measurements were performed using the Gallios flow cytometer (Beckman Coulter). Three biological replicates were analyzed, each with technical triplicates.
For the YUMMER 1.7 D4J cell line analysis the protocol was performed as follows: Acat1-KO and control D4J cells were grown to 70% confluence and scraped using ice-cold PBS. Cell pellets were stained with Live/Dead fixable Red (Invitrogen) followed by surface staining with H2-kb/H2-kd and Pdl1 in FACS buffer (PBS containing 1% FBS, 0.1% sodium azide). For digested tumor samples, single cell suspensions were stained with Live/Dead fixable Red (Thermo Fisher Scientific) in PBS followed by surface staining with antibodies in FACS buffer. For intracellular staining, single cell suspensions were stimulated with PMA (50ng)/Ionomycin (500ng) in the presence of Brefeldin A (BD Biosciences) for 4 h. After fixation and permeabilization, following manufacturer’s recommendations, cells were stained for intracellular cytokines. All samples were acquired on a BD LSRII flow cytometer and analyzed with FlowJo software.
+ Open protocol
+ Expand
2

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
LIVE/DEAD Fixable Red or LIVE/DEAD Fixable Aqua (Invitrogen) staining was performed at 4°C or on ice for 15 min in PBS. Surface Ab staining was performed at 4°C or on ice for 20 min in PBS containing 2% FBS and 2 mM EDTA. For staining intracellular transcription factors, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set Kit prior to intracellular staining for 1 h at 4°C or on ice. Abs for flow cytometry included CD4 (RM4-5; Invitrogen), CD8 (53-6.7; eBioscience), CD25 (PC61; BD Biosciences), CD44 (IM7; eBioscience), CD62L (MEL-14; BD Biosciences), and Foxp3 (FJK-16S; eBioscience). Cells were analyzed or sorted on LSR II, LSRFortessa, and FACSAria II flow cytometers (BD Biosciences) with BD FACSDiva software. Data analysis was performed using FlowJo v.10.2 (Tree Star). FACS data presented in Fig. 3 are gated on forward scatter and side scatter properties indicative of singlet lymphocytes and live CD4+CD8(CD4SP) cells.
+ Open protocol
+ Expand
3

Multiparametric Flow Cytometry Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
LIVE/DEAD Fixable Red or LIVE/DEAD Fixable Aqua (Invitrogen) staining was performed at 4°C or on ice for 15 min in PBS. Surface Ab staining was performed at 4°C or on ice for 20 min in PBS containing 2% FBS and 2 mM EDTA. For staining intracellular transcription factors, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set Kit prior to intracellular staining for 1 h at 4°C or on ice. Abs for flow cytometry included CD4 (RM4-5; Invitrogen), CD8 (53-6.7; eBioscience), CD25 (PC61; BD Biosciences), CD44 (IM7; eBioscience), CD62L (MEL-14; BD Biosciences), and Foxp3 (FJK-16S; eBioscience). Cells were analyzed or sorted on LSR II, LSRFortessa, and FACSAria II flow cytometers (BD Biosciences) with BD FACSDiva software. Data analysis was performed using FlowJo v.10.2 (Tree Star). FACS data presented in Fig. 3 are gated on forward scatter and side scatter properties indicative of singlet lymphocytes and live CD4+CD8(CD4SP) cells.
+ Open protocol
+ Expand
4

Comprehensive Immune Cell Profiling in BALF

Check if the same lab product or an alternative is used in the 5 most similar protocols
The staining panel for immune cell subsets in BALF was designed according to previous studies [44 (link),45 (link)]. Briefly, the BALF cells were stained with fluorochrome-conjugated monoclonal antibodies at 4 °C for 30 min. The antibodies included FITC conjugated anti-Ly6G (1A8; BD Biosciences, San Jose, CA, USA), PE-conjugated anti-Siglec-F (E50-2440; BD Biosciences), APC-conjugated anti-B220 (RA3-6B2; BD Biosciences), APC-conjugated anti-CD3 (145-2C11; BD Biosciences), PerCP/Cy5.5-conjugated anti-CD11b (M1/70; BioLegend, San Diego, CA, USA), eFluor 450-conjugated anti-CD11c (N41B; Invitrogen, Eugene, OR, USA), and Live/Dead fixable Red (Invitrogen). After washing, the samples were analyzed with multi-parametric flow cytometry (LSR II; BD Boisciences) and data were analyzed with FlowJo software (version 10, Tree Star, Inc., Ashland, OR, USA).
+ Open protocol
+ Expand
5

Intestinal T cell Isolation and Characterization

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cryopreserved single-cell suspensions were thawed at 37°C, aliquots of IEL/LPL fraction pooled (when applicable), and rested for 8 hours in RPMI/10% FBS. Single-cell suspensions were incubated with Fc-receptor block for 10 minutes at 4°C (TruStain FcX, BioLegend) followed by hashing antibody (0.5μg per 2 million cells) for 30 minutes at 4°C. Following multiple washes, samples were stained with anti-CD3 (clone OKT3, BioLegend) FITC, anti-CD4 (clone SK3, BD-Horizon) BUV737, anti-CD8α (clone RPA-T8, BioLegend) BV785, anti-CD45 (clone HI30, BioLegend) BV711, and anti-TCRγδ (clone 5A6.E9, Thermo Fisher) PE. Gluten challenge samples were stained with anti-CD38 (clone HIT2, BioLegend) APC, anti-CD103 (clone Ber-ACT8, BioLegend) BV711, and anti-Integrin β7 (clone FIB504, BioLegend) PE/Dazzle 594. Samples were incubated with pre-titrated TotalSeq-C CITE-seq antibody cocktail (BioLegend, PN900000115) for 30 minutes at 4°C. LIVE/DEAD Fixable Red (ThermoFisher) and DAPI were used to gate viable cells. T cells were sorted into RPMI by FACS AriaII or Propel Labs Bigfoot Cell Sorter by gating viable CD3+ intestinal cells and CD3+CD4CD38+CD103+ cells from gluten challenge samples. FACS analysis performed using FlowJo (v.10.7.1) software.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!