Western blot Biotin -NA-

Get tips on using Zombie Aqua™ Fixable Viability Kit to perform Live / Dead assay mammalian cells - human peripheral blood mononuclear cells

Get tips on using STEMdiff™ Trilineage Differentiation Kit to perform Stem cell Differentiation media Differentiation of Human primed induced pluripotent stem cells (UMN PCBC16iPS) into naive pluripotent stem cells

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Tissue - Human Blood / Serum / Plasma / Buffy coat

Get tips on using EBMTM Endothelial Cell Growth Basal Medium, 500 mL to perform 3D Cell Culture Media Human blood-brain barrier organoid

Get tips on using Quant-iT™ RiboGreen™ RNA Assay Kit to perform RNA quantification Fuorimetric - human peripheral blood mononuclear cells (PBMCs)

Get tips on using EBMTM-2 Endothelial Cell Growth Basal Medium-2 to perform Stem cell culture media Cord blood-derived endothelial cells(hCBiPS2)

Get tips on using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit to perform Cell line authentication Human iPSC cells derived from peripheral blood mononuclear cells

Get tips on using Corning® 500 mL MEM (Minimum Essential Medium) Alpha Medium to perform Stem cell culture media Cord blood-derived endothelial cells(hCBiPS2)

Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of the enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.