China
Africa
Explore chapters and articles related to this topic
Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Method: Harvest cells in the usual way. Do not use trypsin to detach adherent cells. Rinse cells with cation-free PBS, and then use ethylenediaminetetraacetic acid (EDTA) in PBS. Wash cells in TCM-N3 by centrifuging in 50 mL conical tubes for 10 min at 2000 rpm. Aspirate supernatant and resuspend in 1 mL TCM-N3. Count the cells using a hemocytometer. The ideal number of cells is approximately 5 × 10 per tube, and less than 5 × 10 is too few. Resuspend in n × mL of TCM-N3, where n is the number of tubes you will have.Pipette 1 mL of the cell suspension into each of your 12 × 75 mm tubes (having labeled them first). Centrifuge for 5 min at 1500 rpm. Aspirate all but approximately 10 µL of the supernatant. Resuspend cells by vortexing or flicking the tube.Dispense the antibodies into microcentrifuge tubes; with multicolor experiments, all the antibodies for one tube can be premixed before addition to cells. Centrifuging briefly in a microcentrifuge removes aggregates and reduces background staining. Add the appropriate amount of antibody to each tube and incubate for 15 min at room temperature.Resuspend cells by flicking the tube or vortexing gently and add 1 mL of DPBS-N3. Centrifuge tubes at 1500 rpm for 5 min. Aspirate supernatant; resuspend cells by flicking or vortexing. Slowly add 0.5 mL of 1% formaldehyde in PBS while vortexing gently. Samples should be left for at least 1 h in the formaldehyde solution before running on the cytometer if they are virus infected or possibly virus infected.Paraformaldehyde is the solid form of polymerized formaldehyde. Formaldehyde solution should be made fresh and not kept for more than a week. The most convenient form is vials of 20% formaldehyde. Old formaldehyde can spoil your data by increasing nonspecific background fluorescence. If you have to make it up from the powdered paraformaldehyde, mix the powder with PBS and leave in a water bath for a day or two. Heating on a hotplate is not recommended, as the fumes are dangerous.A method of removing adherent cells from flasks: Make up PBS with 0.53 mM EDTA. It is important that there are no divalent cations in the PBS, for example, Ca2+ or Mg2+. Wash the monolayer free of tissue culture medium with PBS–EDTA. Decant off fluid and add more PBS–EDTA. Incubate for 15 min at 35°C. Rocking the flask may help. For really stubborn cells, you may need to give the flask a few hard raps to detach them.
Disentangling the H2 E, F(1Σ g +) (v′=0−18)←X(1Σ g +)(v″=3−9)(2+1) REMPI spectrum via 2D velocity-mapped imaging
Published in Molecular Physics, 2021
Mitchell S. Quinn, Klaas Nauta, Scott H. Kable
The vacuum chambers and velocity-mapped imaging (VMI) apparatus used to record the H spectra for this study have been described in detail previously [26, 27]. A few percent seed-ratio molecular beam of HCO in helium was prepared by heating solid paraformaldehyde (Sigma-Aldrich ) to approximately 50C in a small stainless steel sample container just behind the nozzle, while passing 1–2 bar of helium gas through it. Paraformaldehyde is a stable compound of formaldehyde and water, which releases formaldehyde gas upon gentle heating. The mixture was dried in a second container with magnesium sulphate, held at the same temperature as the paraformaldehyde sample. The nozzle (general valve series 9, 0.8 mm orifice) was heated to 70C to prevent recondensation. The expansion passed through a 1 mm skimmer into a second vacuum chamber containing the stack of VMI electrodes [25], coaxially alligned with the molecular beam, which enters the stack through a 6 mm hole in the first electrode (the repeller).