# Passaging Adherent Mammalian Cells > Published on Vicena | [View on web](https://vicena.ai/wiki/passaging-adherent-mammalian-cells-s2BjRA) --- ## User What is the protocol for passaging adherent mammalian cells, including trypsinization time, split ratios for common cell lines like HEK293 and HeLa, and how to count cells with a hemocytometer? --- ## Vicena Passaging (or subculturing) adherent mammalian cells is a critical technique for maintaining cell health and expanding populations. The following protocol details the process for common lines like **HEK293** and **HeLa**, including specific parameters and counting methods. ### 1. General Passaging Protocol The goal of passaging is to detach cells from their current vessel, dissociate them into a single-cell suspension, and seed them into new vessels at a lower density. 1. **Aseptic Preparation:** Pre-warm culture media (e.g., DMEM + 10% FBS) and Trypsin-EDTA to 37°C. 2. **Wash:** Aspirate the old media. Gently wash the cell monolayer with $\ce{Ca^{2+}}$/$\ce{Mg^{2+}}$-free PBS. This is crucial because residual serum contains trypsin inhibitors that will prevent detachment. 3. **Trypsinization:** Add enough Trypsin-EDTA to cover the surface (e.g., 1–2 mL for a 10 cm dish). Incubate at 37°C. 4. **Neutralization:** Once cells have detached (rounded up and floating), add an equal or double volume of complete growth media. The serum in the media inactivates the trypsin. 5. **Dissociation:** Gently pipette the suspension up and down (trituration) to break up cell clumps. 6. **Seeding:** Transfer the required volume of cell suspension into new vessels containing fresh media. ### 2. Cell-Specific Parameters While the steps are similar, the "stickiness" and growth rates of HEK293 and HeLa differ: | Parameter | HEK293 / HEK293T | HeLa | | :--- | :--- | :--- | | **Adherence** | Semi-adherent (very easy to detach) | Strongly adherent | | **Trypsinization Time** | **1–3 minutes**; often detach with gentle tapping even without heat. | **3–5 minutes**; usually requires 37°C incubation. | | **Common Split Ratios** | 1:10 to 1:20 (fast growers) | 1:3 to 1:8 | | **Doubling Time** | ~12–24 hours | ~24 hours | | **Confluency for Split** | 80–90% | 80–90% | *Note: HEK293 cells can easily "slough off" if PBS is added too forcefully. Always pipet liquids down the side of the vessel wall.* ### 3. Cell Counting with a Hemocytometer To ensure reproducible experiments, cells should be counted to determine the exact seeding density (e.g., $2 \times 10^5$ cells/mL). #### **Procedure** 1. **Prepare the Chamber:** Clean the hemocytometer and coverslip with 70% ethanol. Place the coverslip over the counting chambers. 2. **Staining (Viability):** Mix your cell suspension 1:1 with **Trypan Blue** (e.g., 10 µL cells + 10 µL dye). Live cells exclude the dye (clear), while dead cells take it up (blue). 3. **Loading:** Pipette ~10 µL of the mixture into the notch of the hemocytometer. The fluid will be drawn in by capillary action. 4. **Counting:** Under a 10x objective, count the cells in the **4 large corner squares** (each composed of 16 smaller squares). * *Rule:* To avoid double-counting, count cells touching the top and left borders, but ignore those touching the bottom and right borders. #### **Calculations** Use the following formula to find the concentration of your original suspension: $$ \text{Cells/mL} = \left( \frac{\text{Total Cells Counted}}{\text{Number of Squares}} \right) \times \text{Dilution Factor} \times 10^4 $$ * **Dilution Factor:** If you mixed 1:1 with Trypan Blue, the factor is **2**. * **$10^4$:** This constant converts the volume of one square (0.1 mm³) to 1 mL. **Example:** If you counted 200 cells across 4 squares with a 1:1 dilution: $$ \left( \frac{200}{4} \right) \times 2 \times 10^4 = 1 \times 10^6 \text{ cells/mL} $$ ### 4. Summary of Best Practices * **Avoid Over-confluency:** Splitting cells at 100% confluency can lead to contact inhibition and reduced health in future passages. * **Log Passages:** Keep track of the passage number; many lines (especially HEK293) change phenotypically after 20–30 passages. * **Visual Check:** Always verify detachment under a microscope before adding neutralizing media to ensure a high yield. ---