Lumbar punctures (LPs) were performed between the hours of 9 am and 12 pm. An average volume of 20 ml CSF was collected and spun immediately (1200 rpm, 10 min., 4°C). The CSF supernatant was removed from the cell pellet and stored on ice. CSF cells were re-suspended in x-vivo media (Lonza), counted using a Neubauer hemocytometer and distributed among different assays.
Hemocytometer
Manufactured by Lonza
A hemocytometer is a device used for counting and enumerating cells, such as blood cells, in a sample. It consists of a thick glass slide with a precise grid etched into the surface, which is used to determine the concentration of cells in a liquid sample. The grid pattern allows for accurate cell counting and calculation of the total number of cells present in the sample.
Automatically generated - may contain errors
4 protocols using hemocytometer
1
CSF Collection and Processing Protocol
2
Lung Cell Isolation and Quantification
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Lungs were perfused with a phosphate-buffered saline (PBS) solution. Afterward the left lung lobe was collected, minced, and incubated with digestion medium (RPMI medium with 1 mg/ml collagenase type 2) for 45 min at 37°C. Red blood cells were lysed with an ammonium–chloride–potassium (ACK) lysing solution. The total lung viable cell count was determined via a Bruker hemocytometer using Trypan Blue colouring (BioWhittaker® Lonza).
3
Quantifying Cell Populations in BAL Fluid
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Bronchoalveolar lavage (BAL) fluid was obtained by rinsing the lung three times with 750 µl of sterile saline (0.9% NaCl, B. Braun). BAL fluid was centrifuged for 10 min at 1,000 g at 4°C. Total viable cell count was determined via a Bruker hemocytometer using Trypan Blue staining (BioWhittaker® Lonza). Cytospins in duplicate were made and stained with the DiffQuik method (Medical Diagnostics, Germany) for differential cell count. A total of 200 cells were counted to determine the percentage of macrophages, lymphocytes, neutrophils, and eosinophils in BAL fluid.
4
Optimizing Hybridoma Cell Culture Conditions
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Suspension mouse-mouse hybridoma cell line, SP2/0-Ag14 (ATCC, Manassas, VA) were thawed from liquid nitrogen tank and after a few passages were transferred to modified T-75 flasks to study the effectiveness of the method developed for removing leachables and extractables. 21 ml of complete medium (90% v/v of DMEM supplemented with 10% v/v of FBS) were added to each flask. The seeding density in each T-flask was 6.3 × 104 viable cells/mL. Cell density and viability were determined using hemocytometer and trypan blue dye (Lonza, Walkersville, MD) technique.
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